Anti-PD-1 x il-25 bispecific antibody to treat cancer

Abstract

The subject matter described here relates to anti-PD-1 x anti-IL-25 bispecific antibodies. In certain embodiments the bispecific antibody is used for the treatment or prevention of cancer. In certain embodiments the bispecific antibody is used for the treatment or prevention of breast cancer. In some embodiments, the bispecific antibody limits immune related adverse events.

Description

Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 ANTI-PD-1 X IL-25 BISPECIFIC ANTIBODY TO TREAT CANCER

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 649841, filed on May 20, 2024, U.S. Provisional Patent Application No. 63 / 696323, filed on September 18, 2024, U.S. Provisional Patent Application No. 63 / 734994, filed on December 17, 2024, and U.S. Provisional Patent Application No. 63 / 795102, filed on April 25, 2025, which are incorporated herein by reference in their entirety.

[0002] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records but otherwise reserves any and all copyright rights. INCORPORATION BY REFERENCE

[0003] All documents cited herein are incorporated herein by reference in their entireties. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0004] This invention was made with government support under AI125640, AI175498, and AI150597 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0005] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 20, 2025, is named 0019240_01340WO1_SL.xml and is 90,082 bytes in size. TECHNICAL FIELD

[0006] The present invention relates generally to antibodies and methods of treating cancer with said antibodies. More particularly, described herein are methods of simultaneously blocking the receptor PD-1 and IL-25 (IL17E) on cytotoxic T cells with bispecific antibodies and the use of such methods to treat a disease in a subject in need thereof. BACKGROUND

[0007] In 2023, there were 1.9 million new cancer cases in the U.S., leading to over 600,000 deaths. Cancer immunotherapy, using one’s immune system to fight cancer, is at the frontier of cancer treatment. Several types of cancer immunotherapy include immune ActiveUS 209758784 1Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 checkpoint inhibitors (ICI), CAR-T cells, engineering TCR, tumor-infiltrating lymphocytes, and therapeutic cancer vaccines. Among the different types of cancer immunotherapy, ICI is the least invasive, most widely applicable, and most cost-effective. In fact, within the 1.9 million patients newly diagnosed with cancer in 2023, 810,000 patients are eligible for ICI treatments. ICI is considered the new standard of care across many cancer indications. It has improved outcomes and extended patient survival. However, the current response rate to ICIs is only 12.5%, due to primary and acquired resistance. Additionally, ICIs often induce immune-related adverse events (irAEs), some of which, such as myocarditis, could be life- threatening. Moreover, advanced irAEs warrant ICI therapy cessation, limiting the overall effectiveness and usability of this class of potentially lifesaving therapeutic agents.

[0008] Anti-PD-1 antibodies are an essential tool to treat tumors. In some types of tumors, this treatment, alone or in combination, can lead to durable responses. Yet, most patients do not respond, and half develop immune-related adverse events (irAEs). There is a need for prophylactic and / or therapeutic agents that harness the effectiveness of anti-PD-1 immunotherapy but reduce irAEs and / or are more effective in tumor suppression. SUMMARY OF THE INVENTION

[0009] In certain aspects, the present disclosure provides a bispecific antibody or a fragment thereof, comprising: a first arm comprising a first variable heavy chain (VH) domain and a first variable light chain (VL) domain, wherein a portion of the first arm is capable of binding to a portion of an IL-25 protein; and a second arm comprising a second VH domain and a second VL domain, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

[0010] In some embodiments, the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together. In some embodiments, the first arm comprises a linker between the first VH domain and first VL domain. In some embodiments, the second arm comprises a linker between the first VH domain and first VL domain. In some embodiments, the linker is a glycine-serine linker.

[0011] In some embodiments, the first and second arms each further comprise a fragment, crystallizable (Fc) region. In some embodiments, the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa.

[0012] In some embodiments, the first VH domain comprises HCDR1 comprising SEQ ID NO: 5, HCDR2 comprising SEQ ID NO: 7, and HCDR3 comprising SEQ ID NO: 9 and ActiveUS 209758784 2Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 wherein the first VL domain comprises LCDR1 comprising SEQ ID NO: 11, LCDR2 comprising SEQ ID NO: 13, and LCDR3 comprising SEQ ID NO: 15.

[0013] In some embodiments, the second VH domain comprises HCDR1 comprising SEQ ID NO: 28, HCDR2 comprising SEQ ID NO: 29, and HCDR3 comprising SEQ ID NO: 30 and wherein the second VL domain comprises LCDR1 comprising SEQ ID NO: 31, LCDR2 comprising SEQ ID NO: 32, and LCDR3 comprising SEQ ID NO: 33 or wherein the second VH domain comprises HCDR1, HCDR2, and HCDR3 of Figure 10 or Table 1 and wherein the second VL domain comprises LCDR1, LCDR2, and LCDR3 of Figure 10 or Table 3.

[0014] In some embodiments, the first VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 1 and wherein the first VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO: 1. In some embodiments, the second VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79 and wherein the second VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79.

[0015] In some embodiments, the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1 and wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1. In some embodiments, the second VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or SEQ ID NO: 36- 44 or SEQ ID NO: 76, 79 and wherein the second VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79. In some embodiments, the first arm comprises an amino acid sequence comprising SEQ ID NO: 1 and wherein the second arm comprises an amino acid sequence comprising SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79.

[0016] In some embodiments, the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell. In some embodiments, the bispecific antibody is capable of enhancing T cell function. ActiveUS 209758784 3Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0017] In some embodiments, the PD-1 protein is located on a T cell, and wherein the bispecific antibody is capable of preventing the PD-1 protein from binding to a PDL-1 protein or a PDL-2 protein on a tumor cell.

[0018] In some embodiments, the bispecific antibody is capable of causing dephosphorylation of a tail of the PD-1 protein. In some embodiments, the bispecific antibody is capable of inducing a cytokine secretion in a T cell. In some embodiments, the cytokine secretion is a secretion of IL-2.

[0019] In some embodiments, the bispecific antibody does not induce immune-related adverse events. In some embodiments, the bispecific antibody does not induce off-target immune infiltration of one or more untargeted organs.

[0020] In certain aspects, the present disclosure provides a pharmaceutical composition comprising: any of the bispecific antibodies described above; and a pharmaceutically acceptable carrier.

[0021] In certain aspects, the present disclosure provides a method of preventing or treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition described above.

[0022] In some embodiments, the cancer is colorectal cancer, lung cancer, bladder cancer, breast cancer, cervical cancer, kidney cancer, leukemia, Hodgkin lymphoma, non- Hodgkin lymphoma, prostate cancer, skin cancer (e.g., melanoma), head and neck cancer, endometrial cancer, colon cancer, rectal cancer, liver cancer, thyroids cancer, esophageal cancer, renal cell cancer, or a combination thereof. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is lung cancer is small cell lung cancer. In some embodiments, the cancer is lung cancer is non-small cell lung cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is renal cell carcinoma.

[0023] In some embodiments, a tumor of the subject is reduced in volume. In some embodiments, growth of a tumor or cancer cells of the subject is inhibited.

[0024] In some embodiments, immune-related adverse events are treated, reduced, or prevented in the subject. In some embodiments, the immune-related adverse events comprise colitis, diarrhea, rash, pruritis, esophagitis, duodenitis, ileitis, neuritis, arthrhtis, vasculitis, nephritis, adrenal insufficiency, hepatitis, thrombocytopenia, anemia, pneumonitis, thyroiditis, hypophysitis, encephalitis, meningitis, uveitis, mucositis, rash, myocarditis, pericarditis, pancreatitis, colitis, enteritis, or any combination thereof. In some embodiments, off-target immune infiltration of one or more untargeted organs in the subject is reduced or ActiveUS 209758784 4Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 prevented. In some embodiments, CD3+T cells are not detected or are not present at elevated levels in one or more untargeted organs in the subject.

[0025] In some embodiments, treatment with the composition described above has a synergistic effect on reducing a tumor volume, cancer treatment, or inhibiting tumor growth compared to the tumor volume reduction, cancer treatment effect, or tumor growth inhibition exhibited by administering a therapeutic dose of the one or more anti-PD1 antibodies or antigen binding fragment thereof alone or a therapeutic dose of the anti-IL-25 antibody or antigen binding fragment thereof alone.

[0026] In some embodiments, the subject is a human.

[0027] In certain aspects, the present disclosure provides a kit for generating a bispecific antibody or fragment thereof, the kit comprising one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described above.

[0028] In certain aspects, the present disclosure provides a kit for generating a bispecific antibody or fragment thereof, the kit comprising: a first vector comprising a polynucleotide sequence encoding a first arm of the bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a IL-25 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

[0029] In some embodiments, the first vector and the second vector are the same vector. In some embodiments, the first vector and the second vector are two different vectors.

[0030] In some embodiments, the first arm comprises a first VH domain and a first VL domain and the second arm comprises a second VH domain and a second VL domain, wherein a VH domain of the first arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein a first VL domain of the first arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, wherein a VH domain of the second arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or SEQ ID NO: 36- 44 or SEQ ID NO: 76, 79, and wherein a VL domain of the second arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79.

[0031] In certain aspects, the present disclosure provides one or more host cells comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described above. ActiveUS 209758784 5Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0032] In certain aspects, the present disclosure provides one or more host cells comprising: a first vector comprising a polynucleotide sequence encoding a first arm of a bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of an IL-25 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

[0033] In some embodiments, the first vector and the second vector are the same vector. In some embodiments, the first vector and the second vector are two different vectors.

[0034] In some embodiments, the first arm comprises a first VH domain and a first VL domain and the second arm comprises a second VH domain and a second VL domain, wherein the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, wherein a VH domain of the second arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79, and wherein a VL domain of the second arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79.

[0035] In certain aspects, the present disclosure provides a method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells described above under conditions suitable for an expression of the one or more vectors; and recovering the bispecific antibody or fragment thereof.

[0036] In certain aspects, the present disclosure provides a method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells described above under conditions suitable for an expression of the first vector and the second vector; and recovering the bispecific antibody or fragment thereof.

[0037] In certain aspects, the present disclosure provides a composition comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described above.

[0038] In certain aspects, the present disclosure provides a composition comprising: a first vector comprising a polynucleotide sequence encoding a first arm of the bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a IL-25 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein. ActiveUS 209758784 6Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0039] In some embodiments, the first vector and the second vector are the same vector. In some embodiments, the first vector and the second vector are two different vectors.

[0040] In some embodiments, the first arm comprises a first VH domain and a first VL domain, wherein the second arm comprises a second VH domain and a second VL domain, wherein the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, wherein a VH domain of the second arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79, and wherein a VL domain of the second arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79.

[0041] In certain aspects, the present disclosure provides a means for binding: a portion of a IL-25 protein; and a portion of a PD-1 protein. In some embodiments, the means comprises a bispecific antibody or fragment thereof.

[0042] In some embodiments, the bispecific antibody or a fragment thereof comprises: a first arm comprising a first VH domain and a first VL domain, wherein a portion of the first arm is capable of binding to the portion of the IL-25 protein; and a second arm comprising a second VH domain and a second VL domain, wherein a portion of the second arm is capable of binding to the portion of the PD-1 protein.

[0043] In some embodiments, the first VH domain comprises HCDR1 comprising SEQ ID NO: 5, HCDR2 comprising SEQ ID NO: 7, and HCDR3 comprising SEQ ID NO: 9 and wherein the first VL domain comprises LCDR1 comprising SEQ ID NO: 11, LCDR2 comprising SEQ ID NO: 13, and LCDR3 comprising SEQ ID NO: 15. In some embodiments, the second VH domain comprises HCDR1 comprising SEQ ID NO: 28, HCDR2 comprising SEQ ID NO: 29, and HCDR3 comprising SEQ ID NO: 30 and wherein the second VL domain comprises LCDR1 comprising SEQ ID NO: 31, LCDR2 comprising SEQ ID NO: 32, and LCDR3 comprising SEQ ID NO: 33 or wherein the second VH domain comprises HCDR1, HCDR2, and HCDR3 of Figure 10 or Table 1 and wherein the second VL domain comprises LCDR1, LCDR2, and LCDR3 of Figure 10 or Table 3.

[0044] In some embodiments, the first VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 1 and wherein the first VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO: 1. In some embodiments, the second VH domain ActiveUS 209758784 7Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79 and wherein the second VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79.

[0045] In some embodiments, the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, and wherein the portion of the first arm is capable of binding to the portion of the IL-25 protein. In some embodiments, the second VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79, wherein the second VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 24, and wherein the portion of second arm is capable of binding to the portion of the PD-1 protein. In some embodiments, the first arm comprises an amino acid sequence comprising SEQ ID NO: 1, wherein the second arm comprises an amino acid sequence comprising SEQ ID NO: 24 or SEQ ID NO: 36-44 or SEQ ID NO: 76, 79.

[0046] In some embodiments, the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell. In some embodiments, the bispecific antibody is capable of enhancing T cell function.

[0047] In some embodiments, the bispecific antibody is capable of binding to the IL-25 portion of the IL-25 protein and to the PD-1 portion on the PD-1 protein, wherein the IL-25 protein and PD-1 protein are located on a same T cell.

[0048] In some embodiments, the bispecific antibody is capable of disrupting a downstream signaling of a PD-1 mediated response in a T cell. In some embodiments, the bispecific antibody is capable of enhancing T cell function.

[0049] In some embodiments, PD-1 protein is located on a T cell, and wherein the bispecific antibody is capable of preventing the PD-1 protein from binding to a PDL-1 protein or a PDL-2 protein on a tumor cell.

[0050] In some embodiments, the bispecific antibody is capable of causing dephosphorylation of a tail of the PD-1 protein. In some embodiments, the bispecific antibody is capable of inducing a cytokine secretion in a T cell. In some embodiments, the cytokine secretion is a secretion of IL-2. ActiveUS 209758784 8Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0051] In some embodiments, the bispecific antibody does not induce immune-related adverse events. In some embodiments, the bispecific antibody does not induce off-target immune infiltration of one or more untargeted organs. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] The following figures depict illustrative embodiments of the invention. The patent or application file contains at least one drawing executed in color. To conform to the requirements for PCT patent applications, many figures presented herein are black-and-white representations of images originally created in color.

[0053] FIG.1 shows a schematic diagram of the anti-IL-25xPD-1 bispecific antibody.

[0054] FIGS.2A-B show a reduction of tumor volume in the MC38 model by the anti- IL-25xPD-1 bispecific antibody. FIG.2A shows a schematic diagram of the administration regimen of the anti-IL-25xPD-1 bispecific antibody in MC38 tumor-implanted mice. FIG. 2B shows the average MC38 tumor growth in B6 wildtype mice with and without anti-IL- 25xPD-1 bispecific treatment.

[0055] FIGS.3A-B show a dose-dependent reduction of tumor volume in the MC38 model by the anti-IL-25xPD-1 bispecific antibody. FIG.3A shows a schematic diagram of the administration regimen of the anti-IL-25xPD-1 bispecific antibody in the MC38 tumor- implanted mice. FIG.3B shows the average MC38 tumor growth in B6 wildtype mice. Mice were either treated with 50µg, 100µg or 200µg per dose of anti-IL-25xPD-1 antibody or untreated.

[0056] FIGS.4A-B show binding curves to human-PD-1-His-coated plates quantified by ELISA. FIG.4A shows binding curves for anti-PD-1 antibody clones 01, 02, 03, and 07, anti-human PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgG1 isotype control, and blank. FIG.4B shows binding curves for anti-PD-1 antibody clones 09, 51, 55, 79, and 80, anti-human PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgG1 isotype control, and blank. EC50 values were calculated with GraphPad Prism (v10.2.1).

[0057] FIG.5 shows binding of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, and 80 to cell-expressed PD-1 by flow cytometry.

[0058] FIG.6 shows anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, and 80 blocking the binding of rhPD-L2 to cell-expressed PD-1.

[0059] FIG.7 shows IL-2 concentrations determined by ELISA following addition of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 and SEE (Staphylococcal Enterotoxin ActiveUS 209758784 9Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 E) in Jurkat-Raji co-culture compared to no SEE and no antibody control and SEE and no antibody control.

[0060] FIG.8 shows IL-2 concentrations determined by ELISA following addition of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 at different concentrations (10, 2, 0.5 or 0.1 µg / ml) and SEE (Staphylococcal Enterotoxin E) in Jurkat-Raji co-culture compared to no SEE and no antibody control and SEE and no antibody control.

[0061] FIG.9 shows concentrations of IL-2, IFNγ, IL-6, IL-4 and IL-1β determined by ELISA in PBMCs in the presence of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 and SEE (Staphylococcal Enterotoxin E).

[0062] FIG.10 shows anti-PD1 antibody sequences with CDRs highlighted. Figure 10 discloses SEQ ID NOS 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, and 71, respectively, in order of appearance.

[0063] FIG.11 shows Nivolumab / Pembrolizumab bound CD4+ T-cells in cancer patients with irAE-arthritis express high IL-17RA (receptor for IL25 cytokine). T-cells are compared from synovial fluid of irAE-arthritis patients and OA patients by multiparameter flow cytometry. The irAE-arthritis patients express high IL-17RA level in aPD1 bound CD4+ t-cells (cluster 9 and 12).

[0064] FIG.12 shows a schematic of the CEPO1.25 bispecific antibody.

[0065] FIG.13 shows that aPD1xIL25 binds to immobilized murine PD-1 and murine IL-25 by ELISA.

[0066] FIG.14 shows that aPD1xIL25 also binds to human PD-1 on the surface of T cells better than does commercial anti-PD1 antibody, Pembrolizumab.

[0067] FIG.15 shows that aPD1xIL25 regulates cytokine secretion in PBMCs. PBMCs were isolated from 3 different donors using Lymphoprep. Post isolation, PBMCs were incubated overnight in T-cell media. Cells were activated with SEE and treated with nothing, 10µg / ml aPD1xIL25 bispecific antibody, 5µg / ml aPD1+5µg / ml aIL25 antibody, or 5µg / ml aIL25 monoclonal antibody, and cultured for 20 hours. ELISAs were performed on the supernatants of different conditions, and cytokine secretions were measured. The graphs show fold change relative to SEE only no antibody condition.

[0068] FIG.16 shows preliminary testing of aPD1xIL25 in B6 WT mice implanted with colon adenocarcinoma. B6 wildtype females were inoculated with 1,000,000 MC38 tumor cells. At day 4 post inoculation, tumor volumes were approximately 30mm3and treatments were initiated. Per treatment, mice were dosed with nothing or 200µg of aPD1xIL25 ActiveUS 209758784 10Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 bispecific antibody through IP injection. Treatments were given every 3 to 4 days for three times total. At 22 days post tumor inoculation, the experiment was ended, and the mice were euthanized. The tumor growth curve shows the average tumor volume of each experiment group over the course of day 4 to day 22 post tumor cell inoculation.

[0069] FIG.17 shows dose response of aPD1xIL25 in B6 WT mice implanted with colon adenocarcinoma. B6 wildtype females were inoculated with 1,000,000 MC38 tumor cells. At day 7 post inoculation, tumor volumes were approximately 50mm3and treatments were initiated. Per treatment, mice were dosed with nothing, 50µg of aPD1xIL25 bispecific antibody, 100µg of aPD1xIL25 bispecific antibody, or 200µg of aPD1xIL25 bispecific antibody through IP injection. Treatments were given biweekly for two weeks. At 25 days post tumor inoculation, the experiment was ended, and the mice were euthanized. The tumor growth curve shows the average tumor volume of each experiment group over the course of day 7 to day 25 post tumor cell inoculation.

[0070] FIG.18 shows aPDL1xIL25 did not show the same tumor suppressing effect in a similar adenocarcinoma mouse model. B6 Lpr mice were inoculated with 200,000 MC38 tumor cells. When tumor volumes were approximately 20mm3, treatments were initiated. Per treatment, mice were dosed with nothing, 200µg of aPD1 monoclonal antibody, or 200µg of aPDL1xIL25 bispecific antibody through IP injection. Treatments were given biweekly for two and a half weeks. At 15 days post treatment initiation, the experiment was ended, and the mice were euthanized. The tumor growth curve shows the average tumor volume of each experiment group over the course of day 0 to day 15 post treatment initiation.

[0071] FIG.19 shows comprehensive tumor response of aPDL1xIL25 treatment in irAEs susceptible aged B6 Lpr mice. B6 Lpr mice were inoculated with 1,000,000 EO771 tumor cells. When tumor volumes were approximately 50mm3, treatments were initiated. Per treatment, mice were dosed with nothing, 100µg of aPD1 monoclonal antibody, 150µg of aPDL1xIL25 bispecific antibody, or 100µg aPD1+100µg aIL25 antibody (200µg total) through IP injection. Treatments were given biweekly for three weeks. At 22 days post treatment initiation, the experiment was ended, and the mice were euthanized. The tumor growth curve shows the average tumor volume of each experiment group over the course of day 0 to day 22 post treatment initiation. At the endpoint, tumors were collected and weighted. In the bar graph, average tumor mass of each group at 22 days post treatment initiation is illustrated and each dot represents the weight of one primary tumor. ActiveUS 209758784 11Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0072] FIG.20 shows the effect of aPDL1xIL25 treatment on liver and lung immune infiltration in irAEs susceptible aged B6 Lpr mice. B6 Lpr mice were inoculated with 1,000,000 EO771 tumor cells. When tumor volumes were approximately 50mm3, treatments were initiated. Per treatment, mice were dosed with nothing, 100µg of aPD1 monoclonal antibody, or 150µg of aPDL1xIL25 bispecific antibody through IP injection. Treatments were given biweekly for three weeks. At 22 days post treatment initiation, the mice were euthanized. At the endpoint, liver and lung were collected for H&E staining to visualize immune cell infiltration. Immune infiltration severity was graded on a scale of 0-3. The bar graph shows the average immune cell infiltration severity in the liver and lung of mice across different treatment groups. The representative images shows sections of the liver and the lung of individual mice from different treatment groups.

[0073] FIG.21 shows antibody distribution of IgG after aPDL1xIL25 treatment in liver, lung, and tumor in irAEs susceptible aged B6 Lpr mice. B6 Lpr mice were inoculated with 1,000,000 EO771 tumor cells. When tumor volumes were approximately 50mm3, treatments were initiated. Per treatment, mice were dosed with nothing, 100µg of aPD1 monoclonal antibody, or 150µg of aPDL1xIL25 bispecific antibody through IP injection. Treatments were given biweekly for three weeks. At 22 days post treatment initiation, and the mice were euthanized. At the endpoint, liver, lung, and tumor were collected for CD3 staining to visualize T-cell distribution. DAPI staining (blue) shows nuclei of cells. AF647 staining (red) shows cells that are CD3 positive T-cells.

[0074] FIG.22 shows the effect of the bispecific antibody on an In-Vivo Melanoma Model in B6 WT mice. B6 wildtype females were inoculated with 500,000 B16F10 tumor cells. At day 6 post inoculation, tumor volumes were approximately 25mm3and treatments were initiated. Per treatment, mice were dosed with nothing, 100µg aPD1+100µg aIL25 (200µg total), or 150µg of aPD1xIL25 bispecific antibody through IP injection. Treatments were given every 3 days for four times total. At 21 days post tumor inoculation, the mice were euthanized. The tumor growth curve shows the average tumor volume of each experiment group over the course of day 6 to day 21 post tumor cell inoculation.

[0075] FIG.23 shows aPDL1xIL25 did not show the same tumor suppressing effect in an In-Vivo Melanoma Model. B6 wildtype females were inoculated with 500,000 B16F10 tumor cells. At day 6 post inoculation, tumor volumes were approximately 25mm3and treatments were initiated. Per treatment, mice were dosed with nothing or 150µg of aPDL1xIL25 bispecific antibody through IP injection. Treatments were given every 3 days for four times ActiveUS 209758784 12Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 total. At 21 days post tumor inoculation, the mice were euthanized. The tumor growth curve shows the average tumor volume of each experiment group over the course of day 6 to day 21 post tumor cell inoculation.

[0076] FIG.24 shows IL-2 secretion, % of effector CD8+ CD69+ T cells, and CD4+ regulatory T cells in resting cells, activated cells, anti-PD-1 treated cells, anti-PD-1 and anti- IL-25 cotreated cells, and SYB1.25 cells.

[0077] FIG.25 shows IL-6 secretion in resting cells, activated cells, anti-PD-1 treated cells, anti-PD-1 and anti-IL-25 cotreated cells, and SYB1.25 cells.

[0078] FIG.26 shows average MC38 colon tumor volume in B6 mice treated with SYB1.25. Mice are inoculated with MC38 and are untreated, treated with 100 µg per dose, or treated with 200 µg per dose.

[0079] FIGS.27A-B shows average EO771 breast tumor volume in B6 mice treated with SYB1.25. Fig.27A shows that mice are inoculated with EO771 and are untreated, treated with anti-PD-1, or treated with SYB1.25. Fig.27B shows primary tumor mass of untreated, treated with anti-PD-1, or treated with SYB1.25 groups.

[0080] FIG.28 shows average B16F10 melanoma tumor volume in B6 mice treated with SYB1.25. Mice are inoculated with B16F10 and are untreated, treated with anti-PD-1, treated with anti-PD-1 and anti-IL-25, or treated with SYB1.25.

[0081] FIG.29 shows CT26 colon tumor growth in Balb / c mice treated with SYB1.25. Mice are inoculated with CT26 cells and are untreated or treated with SYB1.25. Tumor volume is monitored for 24 days.

[0082] FIG.30 shows CT26 colon tumor rechallenge growth in Balb / c mice treated with SYB1.25. Mice are inoculated with CT26 cells and rechallenged on Day 36. Mice are untreated or treated with SYB1.25. Tumor volume is monitored for 47 days after initial inoculation.

[0083] FIG.31 shows immunohistochemistry of CD3+ T cells in SYB1.25 treated mice. Hematoxylin and eosin staining and CD3+ staining of liver and lung are shown of untreated, anti-PD-1, anti-PD-1+anti-IL-25, and SYB1.25 treated cells.

[0084] FIGS.32A-B shows quantification of severity and CD3+ T cells in SYB1.25 treated mice. FIG.32A shows severity of untreated, anti-PD-1, and SYB1.25 slides. FIG. 32B shows count of CD3+ T cells in untreated, anti-PD-1, anti-PD-1 and anti-IL-25, and SYB1.25 treated cells. ActiveUS 209758784 13Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 DETAILED DESCRIPTION

[0085] As used herein, the term “antibody” includes synthetic antibodies, monoclonal antibodies, oligoclonal or polyclonal antibodies, multiclonal antibodies, recombinantly produced antibodies, intrabodies, monospecific antibodies, monovalent antibodies, multispecific antibodies, multivalent antibodies, bispecific antibodies, bivalent antibodies, human antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies, primatized antibodies, Fab fragments, F(ab’) fragments, F(ab’)2fragments, Fv fragments, single-chain FvFcs (scFv-Fc), single-chain Fvs (scFv), Dabs, nanobodies, anti-idiotypic (anti- Id) antibodies, and any other immunologically-reactive / antigen-binding fragments thereof. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

[0086] The term “bispecific antibody” refers to an antibody having specificities for at least two different, typically non-overlapping, epitopes. Such epitopes may be on the same or different targets. If the epitopes are on different targets, such targets may be on the same cell or different cells or cell types. In some embodiments, the bispecifc antibody comprises a first and second chain. In some embodiments, the first chain comprises an scFv with specificity for a first epitope and the second chain comprises an scFv with specificity for a second epitope. In some embodiments, the first and second chains each further comprise a Fc domain. In some embodiments, the bispecific antibody comprises a first and a second heavy chain. In some embodiments, the bispecific antibody comprises a first and a second heavy chain and a first and a second light chain. In some embodiments, the bispecific antibody comprises any of the designs described in Figure 2 of Brinkmann U, Kontermann RE, The making of bispecific antibodies, MAbs, 2017 Feb / Mar, 9(2):182-212, PMID: 28071970 the content of which is hereby incorporated by reference in its entirety. In some embodiments, at least one of the specificities of the bispecific antibody is for IL-25 and at least one of the specificities is for PD-1. In some embodiments, the bispecific antibody binds to IL-25 and PD-1.

[0087] Disclosed herein are methods for simultaneously targeting IL-25 and PD-1 and the use of such methods to treat or prevent cancer, autoimmunity, or irAEs (immune-related adverse events) in a subject in need thereof. Published experimental data shows that the levels of the cytokine IL-25 are increased in irAE mice and that cancer patient T cells expressed higher levels of the receptor for IL-25. An inverse correlation between cancer patients’ survival and the levels of IL-25 has also been shown and that neutralization of this ActiveUS 209758784 14Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 cytokine, or its receptor, can inhibit tumor growth and prevent irAEs in mice alone and combined with anti-PD-1 antibodies.

[0088] Immune checkpoint inhibitors (ICI) have improved outcomes and extended patient survival in several tumor types. However, ICI often induces immune-related adverse events (irAEs) that warrant therapy cessation, thereby limiting the overall effectiveness of this class of therapeutic agents. Currently, available therapies used to treat irAEs might also blunt the antitumor activity of the ICI themselves. Disclosed herein are novel bispecific antibodies that in some embodiments are useful for preventing or treating cancer. While monoclonal antibodies have been developed to inhibit the immune checkpoint PD-1 on T cells from binding to its ligands, PDL-1 and PDL-2 on cancer cells, these monospecific antibodies lack specificity and can cause adverse events in patients. Described herein are antibodies, including, but not limited to, bispecific antibodies that bind to PD-1 and IL-25. Described herein is a novel anti-PD-1 x anti-IL-25 bispecific antibody upgrading from combination treatment with monospecific antibodies, as a next-generation immune checkpoint inhibitor for cancer. In some embodiments, anti-PD-1 x anti-IL-25 bispecific antibody leads to stronger anti-tumor efficacy and / or less immune toxicity. Described herein are novel anti-PD-1 x anti-IL-25 bispecific antibodies that, in some embodiments, are advantageous over existing anti-PD-1 monospecific antibodies for prevention or treatment of one or more cancers. In some embodiments, the novel bispecific antibodies are useful for preventing or treating autoimmunity. In some embodiments, the novel bispecific antibodies are useful for preventing or treating irAEs. T Cells and PD-1

[0089] T cells have surface receptors that can act as immune checkpoint receptors, such as PD-1. See Speiser DE, Ho PC, Verdeil G. Regulatory Circuits of T Cell Function in Cancer. Nat Rev Immunol.2016 Oct; 16(10): pp.599-611, incorporated by reference in its entirety herein. These receptors act as “checkpoints” to prevent excessive immune activation. However, cancer cells can exploit these checkpoint pathways to evade immune detection and attack. See Liu J, Chen Z, Li Y, Zhao W, Wu J and Zhang Z. PD-1 / PD-L1 Checkpoint Inhibitors in Tumor Immunotherapy. Front. Pharmacol.2021 Sep; pp.12: p.731798, incorporated by reference in its entirety herein. PD-1 is expressed on the surface of activated T cells while its ligands PD-L1 and PD-L2 are expressed on various cancer cells. See Liu, et al. (2021). ActiveUS 209758784 15Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0090] Cancer immunotherapy drugs work by blocking these checkpoint receptors on T cells, thereby unleashing the immune system to recognize and destroy cancer cells more effectively. He X, Xu C. Immune Checkpoint Signaling and Cancer Immunotherapy. Cell Res.2020 Aug; 30(8): pp.660–669, incorporated by reference in its entirety herein. For example, PD-1 inhibitors prevent PD-1 on T cells from binding to its ligands PDL-1 and PDL-2 on tumor cells, thereby preventing the tumor cells from evading the T cell immune response. Seven monoclonal antibodies targeting PD-1 have been approved by the FDA for cancer treatment. However, some individuals do not respond to this treatment, and some experience immune-related adverse events (irAEs).

[0091] Described herein are compositions and methods for preventing or treating cancer. In some embodiments, the composition comprises a bispecific antibody that binds to PD-1 and IL-25.

[0092] In some embodiments, the composition, as disclosed herein, (e.g., composition comprising an anti-PD1 x anti-IL25 bispecific antibody), is used to prevent or treat cancer in a subject. In some embodiments, the cancer is selected from colorectal cancer, lung cancer, bladder cancer, breast cancer, cervical cancer, kidney cancer, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, prostate cancer, skin cancer (e.g., melanoma), head and neck cancer, endometrial cancer, colon cancer, rectal cancer, liver cancer, thyroids cancer, esophageal cancer, renal cell cancer, and a combination thereof. In some embodiments, the cancer is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). In some embodiments, the lung cancer is small cell lung cancer (SCLC). In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is renal cell carcinoma (RCC). In some embodiments, the compositions, as disclosed herein, (e.g., composition comprising an anti-PD1 x anti-IL25 bispecific antibody), is used to prevent or treat disease caused by any solid tumor.

[0093] In some embodiments, described herein are anti-PD1 x anti-IL25 bispecific antibodies. In some embodiments, the anti-PD1 x anti-IL25 bispecific antibodies can be used for cancer immunotherapy. In some embodiments, using one or more of the innovative bispecific antibodies describes herein offer a more potent and safer technology to treat cancer patients resistant to current immunotherapies.

[0094] Described herein are compositions and methods for preventing or treating autoimmunity. In some embodiments, the composition comprises a bispecific antibody that ActiveUS 209758784 16Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 binds to PD-1 and IL-25. In some embodiments, the anti-PD1 x anti-IL25 bispecific antibodies can be used to treat or prevent autoimmunity.

[0095] Described herein are compositions and methods for preventing or treating iRAEs. In some embodiments, the composition comprises a bispecific antibody that binds to PD-1 and IL-25. In some embodiments, the anti-PD1 x anti-IL25 bispecific antibodies can be used to treat or prevent iRAEs. Potentially, any organ can be injured by iRAEs as immune checkpoint inhibitor disrupts self-tolerance to normal tissues. These irAEs range from mild to severe in various tissues, the most common of which include the skin, liver, lung, and gastrointestinal tract. Moderate irAEs require the temporary discontinuation of immune checkpoint inhibitors and short-term use of corticosteroids with subsequent immune checkpoint inhibitor treatment, limiting their efficacy. Severe irAEs often lead to the cessation of life-saving immune checkpoint inhibitor therapy altogether. Other immunosuppressive drugs are usually required, such as high-dose prednisone, methotrexate, tocilizumab, and infliximab. Additionally, prolonged immune suppression may place the patients at risk of developing infections.

[0096] In some embodiments, the subject matter disclosed herein relates to a preventive medical treatment started after following diagnosis of a disease (e.g., cancer) in order to prevent the disease from worsening or curing the disease. In one embodiment, the subject matter disclosed herein relates to prophylaxis of subjects who are believed to be at risk for moderate or severe disease associated with cancer or have previously been diagnosed with another disease, such as cancer. In one embodiment, the subjects can be administered the pharmaceutical composition described herein. The methods of treatment disclosed herein contemplate using any of the antibodies produced by the systems and methods described herein. In one embodiment, the compositions described herein can be administered subcutaneously via syringe or any other suitable method know in the art.

[0097] The compound(s) or combination of compounds disclosed herein, or pharmaceutical compositions (e.g., composition comprising an anti-PD1 x anti-IL25 bispecific antibody) may be administered to a cell, mammal, or human by any suitable means. Non-limiting examples of methods of administration include, among others, (a) administration though oral pathways, which includes administration in capsule, tablet, granule, spray, syrup, or other such forms; (b) administration through non-oral pathways such as intraocular, intranasal, intraauricular, rectal, vaginal, intraurethral, transmucosal, buccal, or transdermal, which includes administration as an aqueous suspension, an oily preparation ActiveUS 209758784 17Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 or the like or as a drip, spray, suppository, salve, ointment or the like; (c) administration via injection, including subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally, intraorbitally, intracapsularly, intraspinally, intrasternally, or the like, including infusion pump delivery; (d) administration locally such as by injection directly in the renal or cardiac area, e.g., by depot implantation; (e) administration topically; as deemed appropriate by those of skill in the art for bringing the compound or combination of compounds disclosed herein into contact with living tissue; (f) administration via inhalation, including through aerosolized, nebulized, and powdered formulations; (g) administration through implantation; and administration via electroporation.

[0098] In some embodiments, one or more antibodies disclosed herein are prepared in a cocktail of DNA-encoding antibodies or mRNA-encoding antibodies and delivered by electroporation to a subject for in vivo expression of the encoded antibodies.

[0099] As will be readily apparent to one skilled in the art, the effective in vivo dose to be administered and the particular mode of administration will vary depending upon the age, weight and species treated, and the specific use for which the compound or combination of compounds disclosed herein are employed. The determination of effective dose levels, that is the dose levels necessary to achieve the desired result, can be accomplished by one skilled in the art using routine pharmacological methods. Typically, human clinical applications of products are commenced at lower dose levels, with dose level being increased until the desired effect is achieved. Alternatively, acceptable in vitro studies can be used to establish useful doses and routes of administration of the compositions identified by the present methods using established pharmacological methods. Effective animal doses from in vivo studies can be converted to appropriate human doses using conversion methods known in the art (e.g., see Nair AB, Jacob S. A simple practice guide for dose conversion between animals and human. Journal of basic and clinical pharmacy.2016 Mar;7(2):27.)

[0100] In some embodiments, the antibodies (or polynucleotides encoding antibodies) prepared using methods of the invention can be combined with additional pharmaceutical components. Antibodies

[0101] There are five classes of human antibodies (i.e., IgA, IgD, IgE, IgG, and IgM) and each have various isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). In some embodiments, the antibodies disclosed herein belong to the IgG class. IgG can be further divided into four subclasses: IgG1, IgG2, IgG3, and IgG4. Each subclass has a unique profile ActiveUS 209758784 18Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 with respect to antigen binding, immune complex formation, complement activation, triggering of effector cells, half-life, and placental transport. E.g., see Gestur Vidarsson, et al., IgG Subclasses and Allotypes: From Structure to Effector Functions, 5 Frontiers in Immunology 520 (2014), incorporated by reference herein in its entirety.

[0102] The IgG immunoglobulin molecule consists of four polypeptide chains, two identical light (L) chains and two identical heavy (H) chains. The four chains are joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region to the dual ends of the “Y”. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each heavy chain consists of an N-terminal variable domain (VH) and three constant domains (CH1, CH2, CH3), with an additional “hinge region” between CH1 and CH2. Similarly, the light chains consist of an N-terminal variable domain (VL) and a constant domain (CL). The variable domains of the heavy chain and light chain may be referred to as “VH” and “VL”, respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). The pairing of a VH and VL together forms a single antigen-binding site. The part of the antibody formed by the lower hinge region and the CH2 / CH3 domains of the heavy chain is called “Fc” (“fragment crystalline”). See e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6, incorporated by reference herein in its entirety.

[0103] The variability in an antibody sequence is concentrated in three segments called complementarity determining regions (CDRs) (also called hypervariable regions (HVRs)) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held together in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site of antibodies. See Kabat et al, Sequences of ActiveUS 209758784 19Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, MD (1991), incorporated by reference in its entirety herein. The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.

[0104] By way of example, CDRs may be defined using the nomenclature described by Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.), incorporated by reference in its entirety herein. Specifically, residues 31- 35 (CDR-H1), 50-65 (CDR-H2), and 95-102 (CDR-H3) in the heavy chain variable region and residues 24-34 (CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in the light chain variable region.

[0105] In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the monoclonal antibody comprises a first, second, third and fourth chain. In some embodiments, the first and third chains each comprise a VH domain and the second and fourth chains each comprise a VL domain. In some embodiments, the first and third chains each further comprises a CH1 domain, a hinge domain, and a Fc domain. In some embodiments, the second and fourth chains each further comprises a CL domain. The pairing of the VH and VL of the first and second chains together forms a single antigen-binding site specific for an epitope on PD-1 and the pairing of the VH and VL of the third and fourth chains together forms a single antigen-binding site specific for an epitope on IL-25. In some embodiments, the first and second chains are linked by one or more covalent disulfide bonds and the third and fourth chains are linked by one or more covalent disulfide bonds. In some embodiments, the first and third chains are linked by one or more disulfide bonds.

[0106] However, the antibodies disclosed herein (e.g., bispecific antibodies) are not limited to full-length antibodies. The antibodies of the various embodiments disclosed herein can include one or more of synthetic antibodies, monoclonal antibodies, oligoclonal or polyclonal antibodies, multiclonal antibodies, recombinantly produced antibodies, intrabodies, monospecific antibodies, monovalent antibodies, multispecific antibodies, multivalent antibodies, bispecific antibodies, bivalent antibodies, human antibodies, humanized antibodies, chimeric antibodies, CDR-grafted antibodies, primatized antibodies, Fab fragments, F(ab’) fragments, F(ab’)2fragments, Fv fragments, single-chain FvFcs (scFv- Fc), single-chain Fvs (scFv), Dabs, nanobodies, anti-idiotypic (anti-Id) antibodies, and any other immunologically-reactive / antigen-binding fragments thereof. These antigen-binding fragments are contemplated herein and a person of skill in the art can readily synthesize such ActiveUS 209758784 20Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 molecules using the sequences and identified domains of the heavy and light chains of the anti-IL-25 and anti-PD-1 antibodies disclosed here. For example, an scFv comprises a variable heavy domain and variable light chain domain separated by a linker. In some embodiments, the linker is a glycine-serine linker. In some embodiments, an antibody can comprise two or more chains each with an Fc domain, wherein the Fc domain of the first chain comprises knob mutations and the Fc domain of the second chain comprise hole mutations, or vice versa. In some embodiments, an antibody comprises the Fc region of IgG1. In some embodiments, an antibody comprises the Fc region of IgG4.

[0107] In some embodiments, the antibody is a bispecific antibody. In some embodiments, the bispecific antibody comprises a first and second chain. In some embodiments, the first chain comprises an scFv with specificity for a first epitope and the second chain comprises an scFv with specificity for a second epitope. In some embodiments, the first and second chains each further comprise a Fc domain. In some embodiments, the bispecific antibody comprises a first and a second heavy chain and a first and a second light chain. The pairing of the first VH and first VL together forms a single antigen-binding site specific for a first epitope and the pairing of the second VH and second VL together forms a single antigen-binding site specific for a second epitope. Such epitopes are on different targets (i.e., PD-1 and IL-25). Such targets may be on the same cell or different cells or cell types. In some embodiments, the bispecific antibody comprises a first and a second heavy chain and does not comprise any light chains, wherein the first VH forms a single antigen- binding site specific for a first epitope and the second VH forms a single antigen-binding site specific for a second epitope. Such epitopes may be on different targets (i.e., PD-1 and IL- 25). Such targets may be on the same cell or different cells or cell types. In some embodiments, the bispecific antibody comprises any of the designs described in Figure 2 of Brinkmann U, Kontermann RE, The making of bispecific antibodies, MAbs, 2017 Feb / Mar, 9(2):182-212, PMID: 28071970 the content of which is hereby incorporated by reference in its entirety.

[0108] In some embodiments, the bispecific antibodies disclosed herein contain various modifications, substitutions, additions, or deletions to the variable or binding regions of one or more arms of an anti-PD-1 x anti-IL-25 antibody disclosed herein. In some embodiments, the monoclonal antibodies, or antigen binding fragments thereof, or the bispecific antibodies disclosed herein may contain substitutions or modifications of the constant region (i.e., the Fc region). In some embodiments, the bispecific antibody comprises the Fc region of IgG1. In ActiveUS 209758784 21Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 some embodiments, the bispecific antibody comprises the Fc region of IgG4. The antibodies disclosed herein may contain one or more additional amino acid residue substitutions, mutations and / or modifications, which result in a compound with preferred characteristics including, but not limited to: altered pharmacokinetics, increased serum half-life, increase binding affinity, reduced binding affinity, reduced immunogenicity, increased production, altered Fc ligand binding, enhanced or reduced ADCC or CDC activity, altered glycosylation and / or disulfide bonds and modified binding specificity. Amino Acid Sequences of Anti-IL-25 Portions of Anti-PD-1 x Anti-IL-25 Bispecific Antibodies

[0109] In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody is a single-chain variable fragment (scFv) with a fragment crystallizable (Fc) region (i.e., scFv-Fc antibody) comprising a variable heavy chain (VH) domain, a linker, a variable light chain (VL) domain, and an Fc portion. Underlined and bolded amino acids represent the respective complementarity determining regions (CDRs). Double underlined amino acids represent a linker sequence between the variable heavy chain and the variable light chain portions. Italicized amino acids represent the Fc portion. Bolded and italicized amino acids represent the “knob” mutations for the production of knob-in-hole antibodies. In some embodiments, instead of the “knob” mutations, the amino acid sequence comprising the anti-IL-25 portion of an anti-PD-1 x anti-IL-25 bispecific antibody can comprise the “hole” mutations, while the “knob” mutations are present on an anti-PD-1 portion of an anti-PD-1 x anti-IL-25 bispecific antibody.

[0110] In some embodiments, the amino acid sequence of the anti-IL-25 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises SEQ ID NO: 1. QVTLKESGPTLVKPTQTLTLTCTFSGFSLNTSGMGVGWIRQPPGKALEWLAHIWWD DVKRYNPALKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARTLPHFFDYWGQ GTTLTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCSASSSVSYMYW YQQKPGKAPKLLIYRTSNLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCKQYHS YPPTWTFGGGTKLEIKRLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITRE PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSD IAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG (SEQ ID NO: 1). In some embodiments, the amino acid sequence of the anti-IL-25 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a ActiveUS 209758784 22Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 signal peptide immediately followed by SEQ ID NO: 1. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0111] In some embodiments, the anti-IL-25 portion (scFv-Fc) of the anti-PD-1 x anti-IL- 25 bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 1. In some embodiments, the anti-IL-25 portion of an anti-PD-1 x anti-IL-25 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody wherein the CDR sequences are indicated above (in bold and underline). In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-IL-25 portion of the anti- PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 1.

[0112] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 2) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length. Nucleic Acid Sequences of Amino Acid Sequences of Anti-IL-25 Portions of Anti-PD-1 x Anti-IL-25 Bispecific Antibodies

[0113] A person skilled in the art can identify the nucleic acid sequences encoding the features identified in the corresponding amino acid sequences (e.g., CDRs, linker sequence, variable heavy chain and variable light chain domains, Fc portion, knob / hole regions, and any amino acids that are associated with reduced binding affinity) by translating the nucleic acid sequences into amino acid sequences. In some embodiments, instead of the “knob” mutations, the nucleic acid sequence comprising the anti-IL-25 portion of an anti-PD-1 x anti-IL-25 bispecific antibody can comprise a nucleic acid sequence encoding the “hole” mutations, while the “knob” mutations are present on an anti-PD-1 portion of an anti-PD-1 x ActiveUS 209758784 23Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 anti-IL-25 bispecific antibody. In some embodiments, the nucleic acid sequences may be codon optimized.

[0114] In some embodiments, the nucleic acid sequence encoding the anti-IL-25 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 comprises SEQ ID NO: 3 CAAGTGACCCTGAAAGAGTCTGGCCCCACACTGGTCAAGCCCACACAGACCCTG ACACTGACCTGCACCTTCAGCGGCTTCAGCCTGAATACCTCTGGCATGGGCGTCG GCTGGATCAGACAGCCTCCTGGAAAAGCCCTGGAATGGCTGGCCCACATTTGGT GGGACGACGTGAAGAGATACAACCCCGCTCTGAAGTCCCGGCTGACCATCACCA AGGACACCAGCAAGAACCAGGTGGTGCTGACCATGACAAACATGGACCCCGTGG ACACCGCCACCTACTACTGTGCTAGAACCCTGCCTCACTTCTTCGACTACTGGGG CCAGGGCACCACACTGACAGTTTCTAGCGGAGGCGGAGGATCTGGTGGCGGAGG AAGTGGCGGAGGCGGTTCTGACATCCAGATGACACAGAGCCCCAGCAGCCTGTC TGCCTCTGTGGGAGACAGAGTGACCATTACCTGTAGCGCCAGCAGCAGCGTGTC CTACATGTACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTAC AGAACCAGCAATCTGGCCAGCGGCGTGCCCAGCAGATTTTCTGGTTCTGGCAGC GGCACCGACTTCACCCTGACCATATCTAGCCTGCAGCCTGAGGACTTCGCCACAT ATTACTGCAAGCAGTACCACAGCTACCCTCCAACCTGGACCTTTGGAGGCGGCAC CAAGCTGGAAATCAAGCGGCTCGAGCCCAAGAGCTGCGACAAGACCCACACCTG TCCTCCATGTCCTGCTCCAGAGTTTCAAGGCGGCCCTTCCGTGTTCCTGTTTCCTC CAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAAGTGACCTGTGTGG TGGTGGATGTGTCCCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTGGACG GCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAACAGC ACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGC AAAGAGTACAAGTGCCAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGAGAAA ACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCTCAGGTGTACGTGTTGCCT CCTAGCAGGGACGAGCTGACCAAGAATCAGGTGTCCCTGCTGTGCCTGGTCAAG GGCTTCTACCCTTCCGACATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTGAG AACAACTACCTGACCTGGCCTCCTGTGCTGGATAGCGACGGCTCATTCTTCCTGT ACAGCAAGCTGACAGTGGACAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCT GCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGAGCC TGTCTCCTGGA (SEQ ID NO: 3). In some embodiments, the nucleic acid sequence encoding the anti-IL-25 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a nucleic acid encoding a signal peptide immediately followed by SEQ ID NO: 3. ActiveUS 209758784 24Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 3 can be determined from the amino acid sequence of SEQ ID NO: 1 as identified herein by a person of skill in the art.

[0115] In some embodiments, the nucleic acid sequence encoding an anti-IL-25 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 3. In some embodiments, the nucleic acid sequence encodes CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-IL- 25 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody wherein the CDR sequences are indicated above (in bold and underline). In some embodiments, the nucleic acid sequence encoding framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-IL-25 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 3.

[0116] Although the sequence above is depicted with a nucleic acid sequence encoding a linker “GGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCT” (SEQ ID NO: 4) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length. In some embodiments, the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length.

[0117] In some embodiments, one or more nucleic acids of a nucleic acid sequence encoding one or more CDRs of SEQ ID NO: 3 is substituted. In some embodiments, one or more nucleic acids of a nucleic acid sequence encoding one or more variable heavy chain CDRs of SEQ ID NO: 3 is substituted. In some embodiments, one or more nucleic acids of a nucleic acid sequence encoding one or more variable light chain CDRs of SEQ ID NO: 3 is substituted. ActiveUS 209758784 25Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 Additional Amino Acid and Nucleic Acid Sequences of An Anti-IL-25 Portion of Anti-PD- 1 x Anti-IL-25 Bispecific Antibodies

[0118] In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises IL-25 binding portion of LNR-125, humanized versions of LNR-125 (also referred to as LNR125.38), and 22C7(Pfizer). In some embodiments, the anti- IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody is a partially or a fully humanized version of LNR-125.

[0119] In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises at least a portion of the amino acid sequence of LNR-125, humanized versions of LNR-125 (also referred to as LNR125.38), 22C7(Pfizer), or any other anti-IL-25 antibody known in the art. In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody is a partially or a fully humanized version of LNR- 125. In some embodiments, the amino acid sequence of the anti-IL-25 portion of the anti- PD-1 x anti-IL-25 bispecific antibody comprises at least a variable heavy and variable light chain portions of the amino acid sequence of LNR-125, humanized versions of LNR-125 (also referred to as LNR125.38), 22C7(Pfizer), or any other anti-IL-25 antibody known in the art. In some embodiments, the amino acid sequence of the anti-IL-25 portion of the anti-PD- 1 x anti-IL-25 bispecific antibody comprises at least the CDRs of the variable heavy chain and the CDRs of the variable light chain portions of the amino acid sequence of LNR-125, humanized versions of LNR-125 (also referred to as LNR125.38), 22C7(Pfizer), or any other anti-IL-25 antibody known in the art. In some embodiments, the nucleic acid sequence the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody codes for an amino acid sequence that comprises at least a portion of the amino acid sequence of LNR-125, humanized versions of LNR-125 (also referred to as LNR125.38), 22C7(Pfizer), or any other anti-IL-25 antibody known in the art. In some embodiments, the nucleic acid sequence of the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody codes for an amino acid sequence that comprises at least a variable heavy and variable light chain portions of the amino acid sequence of LNR-125, humanized versions of LNR-125 (also referred to as LNR125.38), 22C7(Pfizer), or any other anti-IL-25 antibody known in the art. In some embodiments, the nucleic acid sequence encoding the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody codes for an amino acid sequence that comprises at least the CDRs of the variable heavy chain and the CDRs of the variable light chain portions of the amino acid sequence of LNR-125, humanized versions of LNR-125 (also referred to as ActiveUS 209758784 26Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 LNR125.38), 22C7(Pfizer), or any other anti-IL-25 antibody known in the art. The sequences of anti-IL-25 antibodies are described in the art and incorporated herein by reference as follows: LNR-125 (also referred to as ABM125) or an antigen binding fragment thereof as disclosed in the US patent 11,492,397, hereby incorporated in its entirety by reference; 22C7 or an antigen binding fragment thereof disclosed in Bone R et al., Discovery and multi-parametric optimization of a high-affinity antibody against Interleukin-25 with neutralizing activity in a mouse model of skin inflammation, Antibody Therapeutics Vol.5 / Issue 4 pp.258-267 (Oct.2022), hereby incorporated in its entirety by reference.

[0120] In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain with hypervariable regions HCDR1, HCDR2 and HCDR3. HCDR1 comprising the amino acid sequence SEQ ID NO: 5 (TSGMGVG) or the amino acid sequence SEQ ID NO: 6 (SYWIE), HCDR2 comprising the amino acid sequence SEQ ID NO: 7 (HIWWDDVKRYNPALKS) or the amino acid sequence SEQ ID NO: 8 (QILPGIGSTNYNEKFKG), and HCDR3 comprising the amino acid sequence SEQ ID NO: 9 (TLPHFFDY) or the amino acid sequence SEQ ID NO: 10 (GYGNYGDY); or HCDR equivalents thereof. In some embodiments, HCDR1 comprises the amino acid sequence SEQ ID NO: 5, HCDR2 comprises the amino acid sequence SEQ ID NO: 7, and HCDR3 comprises the amino acid sequence SEQ ID NO: 9.. In some embodiments, HCDR1 comprises the amino acid sequence SEQ ID NO: 6, HCDR2 comprises the amino acid sequence SEQ ID NO: 8, and HCDR3 comprises the amino acid sequence SEQ ID NO: 10. In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain which comprises hypervariable regions LCDR1, LCDR2, and LCDR3. LCDR1 comprising the amino acid sequence SEQ ID NO: 11 (SASSSVSYMY) or the amino acid sequence SEQ ID NO: 12 (RASESVDSYGNSFM), LCDR2 comprising the amino acid sequence SEQ ID NO: 13 (RTSNLAS) or the amino acid sequence SEQ ID NO: 14 (RASNLES), and LCDR3 comprising the amino acid sequence SEQ ID NO: 15 (KQYHSYPPTWT) or the amino acid sequence SEQ ID NO: 16 (QQSNEDPLT), or LCDR equivalents thereof. In some embodiments, LCDR1 comprises the amino acid sequence SEQ ID NO: 11, LCDR2 comprises the amino acid sequence SEQ ID NO: 13, and LCDR3 comprises the amino acid sequence SEQ ID NO: 15. In some embodiments, LCDR1 comprises the amino acid sequence SEQ ID NO: 12, LCDR2 comprises the amino acid sequence SEQ ID NO: 14, and LCDR3 comprises the amino acid sequence SEQ ID NO: 16. ActiveUS 209758784 27Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0121] In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 17 (QVTLKESGPTLVKPTQTLTLTCTFSGFSLNTSGMGVGWIRQPPGKALEWLAHIWW DDVKRYNPALKSRLTITKDTSKNQVVLTMTNMDPVDTATYYCARTLPHFFDYWG QGTTLTVSS) or SEQ ID NO: 18 (EVKVVESGADLMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGQILPGIG STNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCARGYGNYGDYWGQGT TVTVSS). In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 19 (DIQMTQSPSSLSASVGDRVTITCSASSSVSYMYWYQQKPGKAPKLLIYRTSNLASGV PSRFSGSGSGTDFTLTISSLQPEDFATYYCKQYHSYPPTWTFGGGTKLEIKRL) or SED ID NO: 20 (DIVLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASN LESGIPARFSGSGSRTDFTLTINPVEADDVATYYCQQSNEDPLTFGAGTKLELKR). In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 17 and comprises a VL domain comprising SEQ ID NO: 19. In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 18 and comprises a VL domain comprising SEQ ID NO: 20. In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 17 or SEQ ID NO: 18 and comprising HCDR1 comprising the amino acid sequence SEQ ID NO: 5 (TSGMGVG) or SEQ ID NO: 6 (SYWIE), HCDR2 comprising the amino acid sequence SEQ ID NO: 7 (HIWWDDVKRYNPALKS) or SEQ ID NO: 8 (QILPGIGSTNYNEKFKG), and HCDR3 comprising the amino acid sequence SEQ ID NO: 9 (TLPHFFDY) or SEQ ID NO: 10 (GYGNYGDY). In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 17 and comprising HCDR1 comprising the amino acid sequence SEQ ID NO: 5, ActiveUS 209758784 28Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 HCDR2 comprising the amino acid sequence SEQ ID NO: 7, and HCDR3 having the amino acid sequence SEQ ID NO: 9. In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 18 and comprising HCDR1 comprising the amino acid sequence SEQ ID NO: 6, HCDR2 comprising the amino acid sequence SEQ ID NO: 8, and HCDR3 having the amino acid sequence SEQ ID NO: 10. In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 19 or 20. In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 19 or 20 and comprising LCDR1 comprising the amino acid sequence SEQ ID NO: 11 (SASSSVSYMY) or SEQ ID NO: 12 (RASESVDSYGNSFM), LCDR2 comprising the amino acid sequence SEQ ID NO: 13 (RTSNLAS) or SEQ ID NO: 14 (RASNLES), and LCDR3 comprising the amino acid sequence SEQ ID NO: 15 (KQYHSYPPTWT) or SEQ ID NO: 16 (QQSNEDPLT). In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 19 and comprising LCDR1 comprising the amino acid sequence SEQ ID NO: 11, LCDR2 comprising the amino acid sequence SEQ ID NO: 13, and LCDR3 comprising the amino acid sequence SEQ ID NO: 15. In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 20 and comprising LCDR1 comprising the amino acid sequence SEQ ID NO: 12, LCDR2 comprising the amino acid sequence SEQ ID NO: 14, and LCDR3 comprising the amino acid sequence SEQ ID NO: 16.

[0122] In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a humanized version of LNR-125 or an antigen binding fragment thereof, as disclosed in the US patent 11,492,397, hereby incorporated in its entirety by reference. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′, ActiveUS 209758784 29Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 F(ab′)2, or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some embodiments, the anti-IL-25 antibody is fully humanized wherein all the framework residues are derived from human immunoglobulins (recipient antibody). In some embodiments, the anti-IL-25 antibody is partially humanized. In some instances, framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for humanizing non-human antibodies are well known in the art.

[0123] In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 21 (QVQLVQSGAEVKKPGASVKVSCKASGYTFSSYWIEWVRQAPGQGLEWIGQILPGIG STNYNEKFKGRVTITADTSTSTVYMELSSLRSEDTAVYYCARGYGNYGDYWGQGTT VTVSS) and a VL domain comprising SEQ ID NO: 22 (DIVLTQSPASLAVSPGQRATITCRASESVDSYGNSFMHWYQQKPGQPPKLLIYRASN LESGVPARFSGSGSGTDFTLTINPVEAQDTANYYCQQSNEDPLTFGAGTKLELKR). In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 21. In some embodiments, the anti-IL-25 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 22. ActiveUS 209758784 30Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 Amino Acid Sequences of Anti-PD-1 Portions of Anti-PD-1 x Anti-IL-25 Bispecific Antibodies

[0124] In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a single-chain variable fragment (scFv) with a fragment crystallizable (Fc) region (i.e., scFv-Fc antibody) comprising a variable heavy chain domain, a linker, a variable light chain domain, and an Fc portion. Underlined and bolded amino acids represent the respective complementarity determining regions (CDRs) or are shown in Figure 10. Double underlined amino acids represent a linker sequence between the variable heavy chain and the variable light chain portions. Italicized amino acids represent the Fc portion. Italicized and double underlined amino acids represent the hinge region. Bolded and italicized amino acids represent the “hole” mutations for the production of knob-in-hole antibodies. In some embodiments, instead of the “hole” mutations, the amino acid sequence comprising the anti-PD-1 portion of an anti-PD-1 x anti-IL-25 bispecific antibody can comprise the “knob” mutations, while the “hole” mutations are present on an anti-IL-25 portion of an anti-PD-1 x anti-IL-25 bispecific antibody.

[0125] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 24: QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSG GTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMV TVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGATYL YWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQ LTHWPYTFGQGTKLEIKRLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITRE PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG (SEQ ID NO: 24). In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., MGWSCIILFLVATATGVHS (SEQ ID NO: 23)) immediately followed by SEQ ID NO: 24. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.

[0126] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises ActiveUS 209758784 31Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 SEQ ID NO: 36: EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSS TIKYTPSLKDKFIISRDNAKNTLCLQLSKVRSEDSALYYCARMGYRLFDSWGQGTTL TVSSGGGGSGGGGSGGGGSNIVMTQATPSVPVTPGESVSISCRSSKSLLHSDGDTYLY WFLQRPGQSPQLLIYRMSNLVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQH LEYPYTFGGGTKLEIKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK EYKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG (SEQ ID NO: 36). In some embodiments, the amino acid sequence of the anti- PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., MGWSCIILFLVATATGVHS (SEQ ID NO: 23)) immediately followed by SEQ ID NO: 36. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.

[0127] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 37: EVQLQQSGTVLARPGASVKMSCKASGNTFTSYWMHWVKQRPGQGLEWIGAIYPGY SDTRYNQKFKGKATLTAVTSTSTAYMELSSLTNEDSAVYYCTKFIATVGGYFDVWG AGTTVTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSSSLGERVTMTCTASSSVSSSYL HWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQ YHRSPPIFTFGSGTKLEIKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITRE PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG (SEQ ID NO: 37). In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., SEQ ID NO: 23) immediately followed by SEQ ID NO: 37. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0128] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 38: ActiveUS 209758784 32Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWIKIETG NPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDYYGYYYYAMDYW GQGTSVTVSSGGGGSGGGGSGGGGSQIVLTQSPAIMSASLGERVTMTCTVSSSISSSY LHWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCH QYHRSPLTFGAGTKLELKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITRE PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG (SEQ ID NO: 38). In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., SEQ ID NO: 23) immediately followed by SEQ ID NO: 26. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0129] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 39: EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYGMSWVRQTPDKRLELVATIDNNGG NTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARDAYYTYDYWYFDV WGAGTTVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCSASQGISN YLNWYQQKPDGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIATYYCQ QYHKLPWTFGGGTKLEIKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITR EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG (SEQ ID NO: 39). In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., SEQ ID NO: 23) immediately followed by SEQ ID NO: 39. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0130] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 40: EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVAYISYGGG ActiveUS 209758784 33Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 DTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQKVDGYFVAMDY WSQGTSVTVSSGGGGSGGGGSGGGGSVIVLTQSPASLAVSLGQRATISCRASERVDD YGISFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTA MYFCQQNKEVPWTFGGGTKLEIKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKD TLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG (SEQ ID NO: 40). In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., SEQ ID NO: 23) immediately followed by SEQ ID NO: 40. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0131] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 41: EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVATISGGGR YTYYPDSVKGRFTISRDNAKNILYLQMSSLRSEDTALYYCASPYDGYYGAMDYWG QGTSVTVSSGGGGSGGGGSGGGGSDIVLTQSPASLAVSLGQRATISCRASESVDNSGI SFMNWFQQKPGQPPKLLIYAASNQGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYF CQQSKEVPWTFGGGTKLEIKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYI TREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG (SEQ ID NO: 41). In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., SEQ ID NO: 23) immediately followed by SEQ ID NO: 41. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0132] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 76: EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISGGG RYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYDGYYGAMDY ActiveUS 209758784 34Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 WGQGTSVTVSSGGGGSGGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASESVDN SGISFMNWYQQKPGQAPRLLIYAASNQGSGIPARFSGSGSGTDFTLTISSLEPEDFAV YYCQQSKEVPWTFGGGTKLEIKRLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPK DTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPG (SEQ ID NO: 76). SEQ ID NO: 76 comprises a humanized version of the VH and VL portions of SEQ ID NO: 41. In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., SEQ ID NO: 23) immediately followed by SEQ ID NO: 76. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0133] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 42: EVQLQQSGTVLARPGASVRMSCKASGYSFSTYWMHWVKQRPGQGLEWIGGIYPGN SDTNYNQKFKGKAKLTAVTSASTAYMDLSSLTNEDSAVYYCTGGYFFDVWGAGTT VTVSSGGGGSGGGGSGGGGSDIKMTQSPSSLYASLGERVTITCKASQDINRYLTWFQ QKPGKSPKTLIFRANRLVTGVPSRFIGSGSGQEYSLTISSLEYEDMGIYFCLQYDESPY TFGGGTKLEIKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQ VSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG (SEQ ID NO: 42). In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., SEQ ID NO: 23) immediately followed by SEQ ID NO: 42. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0134] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 43: QIQLQQSGPEQVKPGASVKISCKASGYMFIDYYMNWVKQKPGQGLEWIGWIYPGSG STKDNENFKGKATLTVDTSSSTAYMQLSSLTSEDTAVYFCARYGPRFFDVWGAGTT VTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASLGERVNLTCRASQEISGYLSWLQ ActiveUS 209758784 35Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 QKPDGTIKRLIYVASTLDSGVPERFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPY TFGGGTKLEIKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQ VSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG (SEQ ID NO: 43). In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., SEQ ID NO: 23) immediately followed by SEQ ID NO: 43. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0135] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 44: EVKLLESGGGLVQPGGSLKLSCEVSGFDFSRDWMSWVRQAPGKGLEWIGQISPDSTS INYKPSLKDKFIISRDNAKNTLYLQLSGVRSEDTALYHCARDTSGYPDYWGQGTSLT VSSGGGGSGGGGSGGGGSDILLTQSPSSMSVSLGDTVSITCHASQDISRNIGWLRQKP GKSFKGLIYHGTNLEDGVPSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPYTF GGGTKLEIKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYITREPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCQVS NKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP G (SEQ ID NO: 44). In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., SEQ ID NO: 23) immediately followed by SEQ ID NO: 44. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0136] In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv- Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 79: EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVAYISYGGG DTYYSDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQKVDGYYVGMDY WGQGSSVTVSSGGGGSGGGGSGGGGSNIVMTQSPKSMSMSVGERVTLSCKASENV GTNVSWYQQKPEQSPRLLIYGASNRYTGVPDRFTGSGSVTDFTLTISSVQAEDLVDY HCGQSYSSPLTFGSGTKLELKLEPKSCDKTHTCPPCPAPEFQGGPSVFLFPPKPKDTLYI TREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD ActiveUS 209758784 36Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 WLNGKEYKCQVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG (SEQ ID NO: 79). In some embodiments, the amino acid sequence of the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide (e.g., MGWSCIILFLVATATGVHS (SEQ ID NO: 23)) immediately followed by SEQ ID NO: 79. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.

[0137] In some embodiments, the signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 23. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 24. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 36. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 37. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 38. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 39. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 40. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 41. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, ActiveUS 209758784 37Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 42. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 43. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 44. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 76. In some embodiments, the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 79. In some embodiments, the anti-PD-1 portion of an anti-PD-1 x anti-IL-25 bispecific antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody wherein the CDR sequences are indicated above (in bold and underline) or in Figure 10 or Tables 1 and 3. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 24. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 36. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 37. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, ActiveUS 209758784 38Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 38. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 39. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 40. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 41. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 42. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 43. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 44. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 76. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 79. In some embodiments, the ActiveUS 209758784 39Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of Tables 2 and 4.

[0138] Although the sequence above is depicted with a linker “GGGGSGGGGSGGGGS” (SEQ ID NO: 2) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker is between about 2 and 25 amino acids in length. In some embodiments, the glycine-serine linker is about 15 amino acids in length.

[0139] In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain with hypervariable regions HCDR1, HCDR2 and HCDR3 sequences disclosed as in either Figure 10 or in Table 1, including combinations thereof (e.g. any HCDR1, HCDR2, HCDR3 of Figure 10 or Table 1 in combination with any HCDR1, HCDR2, HCDR3 of Figure 10 or Table 1) . In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a FR region which comprises HFR regions HFR1, HFR2, HFR3, and HFR4 sequences disclosed in Figure 10 or Table 2. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain which comprises hypervariable regions LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences disclosed in Figure 10 or Table 3, including combinations thereof (e.g. any LCDR1, LCDR2, LCDR3 of Figure 10 or Table 1 in combination with any LCDR1, LCDR2, LCDR3 of Figure 10 or Table 1). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a FR region which comprises LFR regions LFR1, LFR2, LFR3, and LFR4 sequences disclosed in Figure 10 or Table 4.

[0140] Table 1- Alternative HCDR Sequences for anti-PD1 portion ActiveUS 209758784 40Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025ActiveUS 209758784 41Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0141] Table 2- Alternative HFR Sequences for anti-PD1 portion ActiveUS 209758784 42Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025ActiveUS 209758784 43Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0142] Table 3- Alternative LCDR Sequences for anti-PD1 portion ActiveUS 209758784 44Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025ActiveUS 209758784 45Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0143] Table 4- Alternative LFR Sequences for the anti-PD1 portion ActiveUS 209758784 46Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025ActiveUS 209758784 47Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025Nucleic Acid Sequences of Anti-PD-1 Portions of Anti-IL-25xPD-1

[0144] A person skilled in the art can identify the nucleic acid sequences encoding the features identified in the corresponding amino acid sequences (e.g., CDRs, linker sequence, variable heavy chain and variable light chain domains, Fc portion, knob / hole regions, and any amino acids that are associated with reduced binding affinity) by translating the nucleic acid sequences into amino acid sequences. In some embodiments, instead of the “knob” mutations, the nucleic acid sequence comprising the anti-PD-1 portion of an anti-PD-1 x anti- IL-25 bispecific antibody can comprise the “hole” mutations, while the “knob” mutations are present on an anti-IL-25 portion of an anti-PD-1 x anti-IL-25 bispecific antibody. In some embodiments, the nucleic acid sequences may be codon optimized.

[0145] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACA GC (SEQ ID NO: 25). In some embodiments, the nucleic acid sequence encoding the anti- PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 26. CAGGTTCAGCTGGTTCAGTCTGGCGCCGAAGTGAAGAAACCTGGCAGCAGCGTG AAGGTGTCCTGCAAGGCCAGCGGCTTCACCTTCACCACCTACTACATCAGCTGGG TCCGACAGGCCCCTGGACAGGGACTTGAGTACCTGGGCTACATCAACATGGGCA GCGGCGGCACCAACTACAACGAGAAGTTCAAGGGCAGAGTGACCATCACCGCCG ACAAGAGCACAAGCACCGCCTACATGGAACTGAGCAGCCTGAGAAGCGAGGAC ActiveUS 209758784 48Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 ACCGCCGTGTACTACTGTGCCATCATCGGCTACTTCGACTACTGGGGCCAGGGCA CCATGGTCACAGTTTCTAGCGGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCG GAGGCGGTTCTGATGTGGTTATGACACAGAGCCCTCTGAGCCTGCCTGTGACACT GGGACAGCCTGCCAGCATCAGCTGTAGAAGCAGCCAGAGCCTGCTGGATTCTGA CGGCGCCACATACCTGTACTGGTTCCAGCAAAGACCCGGACAGAGCCCCAGACG GCTGATCTACCTGGTGTCTACACTCGGCAGCGGAGTGCCCGATAGATTTTCTGGC AGCGGAAGCGGCACCGACTTCACCCTGAAGATCAGCAGAGTGGAAGCCGAGGA CGTGGGCGTGTACTATTGCATGCAGCTGACCCACTGGCCTTACACCTTTGGCCAG GGGACCAAGCTGGAAATCAAGaggCTCGAGCCCAAGAGCTGCGACAAGACCCAC ACCTGTCCTCCATGTCCTGCTCCAGAGTTTCAAGGCGGCCCTTCCGTGTTCCTGTT TCCTCCAAAGCCTAAGGACACCCTGTACATCACCCGCGAGCCTGAAGTGACCTGT GTGGTGGTGGATGTGTCCCACGAGGACCCCGAAGTGAAGTTCAATTGGTACGTG GACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGAACAGTACAA CAGCACCTACAGAGTGGTGTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAA CGGCAAAGAGTACAAGTGCCAGGTGTCCAACAAGGCCCTGCCTGCTCCTATCGA GAAAACCATCAGCAAGGCCAAGGGCCAGCCTAGGGAACCTCAAGTGTACGTGTA CCCTCCTAGCCGGGACGAGCTGACCAAGAATCAGGTGTCCCTGACCTGCCTCGTG AAGGGCTTCTACCCTTCCGACATCGCCGTGGAATGGGAGAGCAATGGCCAGCCT GAGAACAACTACAAGACAACCCCTCCTGTGCTGGACAGCGACGGCTCTTTTGCCC TGGTGTCCAAGCTGACAGTGGACAAGTCCAGATGGCAGCAGGGCAACGTGTTCA GCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGA GCCTGTCTCCTGGA (SEQ ID NO: 26). In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a nucleic acid sequence encoding a signal peptide (e.g., SEQ ID NO: 25)) immediately followed by SEQ ID NO: 26. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 26 can be determined from the amino acid sequence of SEQ ID NO: 24 as identified herein by a person of skill in the art.

[0146] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACA ActiveUS 209758784 49Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 GC (SEQ ID NO: 25). In some embodiments, the nucleic acid sequence encoding the anti- PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 45. GAAGTGAAACTGCTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTG AAACTGAGCTGCGCGGCGAGCGGCTTTGATTTTAGCCGCTATTGGATGAGCTGGG TGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGATTGGCGAAATTAACCCGGATA GCAGCACCATTAAATATACCCCGAGCCTGAAAGATAAATTTATTATTAGCCGCGA TAACGCGAAAAACACCCTGTGCCTGCAGCTGAGCAAAGTGCGCAGCGAAGATAG CGCGCTGTATTATTGCGCGCGCATGGGCTATCGCCTGTTTGATAGCTGGGGCCAG GGCACCACCCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAG CGGCGGCGGCGGCAGCAACATTGTGATGACCCAGGCGACCCCGAGCGTGCCGGT GACCCCGGGCGAAAGCGTGAGCATTAGCTGCCGCAGCAGCAAAAGCCTGCTGCA TAGCGATGGCGATACCTATCTGTATTGGTTTCTGCAGCGCCCGGGCCAGAGCCCG CAGCTGCTGATTTATCGCATGAGCAACCTGGTGAGCGGCGTGCCGGATCGCTTTA GCGGCAGCGGCAGCGGCACCGCGTTTACCCTGCGCATTAGCCGCGTGGAAGCGG AAGATGTGGGCGTGTATTATTGCATGCAGCATCTGGAATATCCGTATACCTTTGG CGGCGGCACCAAACTGGAAATTAAACTGGAACCGAAAAGCTGCGATAAAACCCA TACCTGCCCGCCGTGCCCGGCGCCGGAATTTCAGGGCGGCCCGAGCGTGTTTCTG TTTCCGCCGAAACCGAAAGATACCCTGTATATTACCCGCGAACCGGAAGTGACCT GCGTGGTGGTGGATGTGAGCCATGAAGATCCGGAAGTGAAATTTAACTGGTATG TGGATGGCGTGGAAGTGCATAACGCGAAAACCAAACCGCGCGAAGAACAGTAT AACAGCACCTATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTG AACGGCAAAGAATATAAATGCCAGGTGAGCAACAAAGCGCTGCCGGCGCCGATT GAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACCGCAGGTGTATGT GTATCCGCCGAGCCGCGATGAACTGACCAAAAACCAGGTGAGCCTGACCTGCCT GGTGAAAGGCTTTTATCCGAGCGATATTGCGGTGGAATGGGAAAGCAACGGCCA GCCGGAAAACAACTATAAAACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTT TGCGCTGGTGAGCAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGT GTTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAG CCTGAGCCTGAGCCCGGGC (SEQ ID NO: 45). In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a nucleic acid sequence encoding a signal peptide (e.g., SEQ ID NO: 25) immediately followed by SEQ ID NO: 45. In some embodiments, the signal peptide is ActiveUS 209758784 50Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 cleaved during post-translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 45 can be determined from the amino acid sequence of SEQ ID NO: 36 as identified herein by a person of skill in the art. In some embodiments, the nucleic acid sequence encoding anti-PD-1 VH portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 82: GAGGTGAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTG AAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTAGATACTGGATGAGTTGGG TCCGGCAGGCTCCAGGGAAAGGGCTAGAATGGATTGGAGAAATTAATCCAGATA GCAGTACGATAAAATACACGCCATCTCTAAAGGATAAATTCATCATCTCCAGAG ACAACGCCAAAAATACGCTGTGCCTGCAATTGAGCAAAGTGAGATCTGAGGACT CAGCCCTTTATTACTGTGCAAGAATGGGCTATAGGCTCTTTGACTCCTGGGGCCA AGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO: 82). In some embodiments, the nucleic acid sequence encoding anti-PD-1 VL portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 83: AATATTGTGATGACTCAGGCTACACCCTCTGTACCTGTCACTCCTGGAGAGTCAG TATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTGATGGCGACACTTA CTTGTATTGGTTCCTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGG ATGTCCAACCTTGTCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGA ACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATT ACTGTATGCAACATCTAGAATATCCGTACACGTTCGGAGGGGGGACCAAGCTGG AAATAAAA (SEQ ID NO: 83).

[0147] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 46. GAAGTGCAGCTGCAGCAGAGCGGCACCGTGCTGGCGCGCCCGGGCGCGAGCGTG AAAATGAGCTGCAAAGCGAGCGGCAACACCTTTACCAGCTATTGGATGCATTGG GTGAAACAGCGCCCGGGCCAGGGCCTGGAATGGATTGGCGCGATTTATCCGGGC TATAGCGATACCCGCTATAACCAGAAATTTAAAGGCAAAGCGACCCTGACCGCG GTGACCAGCACCAGCACCGCGTATATGGAACTGAGCAGCCTGACCAACGAAGAT AGCGCGGTGTATTATTGCACCAAATTTATTGCGACCGTGGGCGGCTATTTTGATG ActiveUS 209758784 51Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 TGTGGGGCGCGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCCAGATTGTGCTGACCCAGAGCCCGGCG ATTATGAGCAGCAGCCTGGGCGAACGCGTGACCATGACCTGCACCGCGAGCAGC AGCGTGAGCAGCAGCTATCTGCATTGGTATCAGCAGAAACCGGGCAGCAGCCCG AAACTGTGGATTTATAGCACCAGCAACCTGGCGAGCGGCGTGCCGGCGCGCTTT AGCGGCAGCGGCAGCGGCACCAGCTATAGCCTGACCATTAGCAGCATGGAAGCG GAAGATGCGGCGACCTATTATTGCCATCAGTATCATCGCAGCCCGCCGATTTTTA CCTTTGGCAGCGGCACCAAACTGGAAATTAAACTGGAACCGAAAAGCTGCGATA AAACCCATACCTGCCCGCCGTGCCCGGCGCCGGAATTTCAGGGCGGCCCGAGCG TGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGTATATTACCCGCGAACCGGA AGTGACCTGCGTGGTGGTGGATGTGAGCCATGAAGATCCGGAAGTGAAATTTAA CTGGTATGTGGATGGCGTGGAAGTGCATAACGCGAAAACCAAACCGCGCGAAGA ACAGTATAACAGCACCTATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGA TTGGCTGAACGGCAAAGAATATAAATGCCAGGTGAGCAACAAAGCGCTGCCGGC GCCGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACCGCAGG TGTATGTGTATCCGCCGAGCCGCGATGAACTGACCAAAAACCAGGTGAGCCTGA CCTGCCTGGTGAAAGGCTTTTATCCGAGCGATATTGCGGTGGAATGGGAAAGCA ACGGCCAGCCGGAAAACAACTATAAAACCACCCCGCCGGTGCTGGATAGCGATG GCAGCTTTGCGCTGGTGAGCAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGG GCAACGTGTTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCA GAAAAGCCTGAGCCTGAGCCCGGGC (SEQ ID NO: 46). In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a nucleic acid sequence encoding a signal peptide (e.g., SEQ ID NO: 25) immediately followed by SEQ ID NO: 46. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 46 can be determined from the amino acid sequence of SEQ ID NO: 25 as identified herein by a person of skill in the art. In some embodiments, the nucleic acid sequence encoding anti-PD-1 VH portion of an anti-PD- 1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 84: GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCAGTG AAGATGTCCTGCAAGGCTTCTGGCAACACCTTTACCAGCTACTGGATGCACTGGG TAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGCGCTATTTATCCTGGAT ActiveUS 209758784 52Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 ATAGTGATACTAGGTACAATCAGAAGTTTAAGGGCAAGGCCACACTGACTGCAG TCACATCCACCAGCACTGCCTACATGGAGCTCAGCAGCCTGACAAATGAGGACT CTGCGGTCTATTACTGTACAAAATTTATTGCTACGGTAGGGGGGTACTTCGATGT CTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 84). In some embodiments, the nucleic acid sequence encoding anti-PD-1 VL portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 85: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTTCATCTCTAGGGGAACGGG TCACCATGACCTGCACTGCCAGCTCAAGTGTAAGTTCCAGTTACTTGCACTGGTA CCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTATAGCACATCCAACCTG GCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTC TCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCAGTA TCATCGTTCCCCACCCATATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA (SEQ ID NO: 85).

[0148] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 47. CAGATTCAGCTGGTGCAGAGCGGCCCGGAACTGAAAAAACCGGGCGAAACCGTG AAAATTAGCTGCAAAGCGAGCGGCTATACCTTTACCGATTATAGCATGCATTGGG TGAAACAGGCGCCGGGCAAAGGCCTGAAATGGATGGGCTGGATTAAAATTGAAA CCGGCAACCCGACCTATGCGGATGATTTTAAAGGCCGCTTTGCGTTTAGCCTGGA AACCAGCGCGAGCACCGCGTATCTGCAGATTAACAACCTGAAAAACGAAGATAC CGCGACCTATTTTTGCGCGCGCGATTATTATGGCTATTATTATTATGCGATGGATT ATTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCCAGATTGTGCTGACCCAGAGCCCGGCG ATTATGAGCGCGAGCCTGGGCGAACGCGTGACCATGACCTGCACCGTGAGCAGC AGCATTAGCAGCAGCTATCTGCATTGGTATCAGCAGAAACCGGGCAGCAGCCCG AAACTGTGGATTTATAGCACCAGCAACCTGGCGAGCGGCGTGCCGGCGCGCTTT AGCGGCAGCGGCAGCGGCACCAGCTATAGCCTGACCATTAGCAGCATGGAAGCG GAAGATGCGGCGACCTATTATTGCCATCAGTATCATCGCAGCCCGCTGACCTTTG GCGCGGGCACCAAACTGGAACTGAAACTGGAACCGAAAAGCTGCGATAAAACC CATACCTGCCCGCCGTGCCCGGCGCCGGAATTTCAGGGCGGCCCGAGCGTGTTTC ActiveUS 209758784 53Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 TGTTTCCGCCGAAACCGAAAGATACCCTGTATATTACCCGCGAACCGGAAGTGA CCTGCGTGGTGGTGGATGTGAGCCATGAAGATCCGGAAGTGAAATTTAACTGGT ATGTGGATGGCGTGGAAGTGCATAACGCGAAAACCAAACCGCGCGAAGAACAG TATAACAGCACCTATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGC TGAACGGCAAAGAATATAAATGCCAGGTGAGCAACAAAGCGCTGCCGGCGCCG ATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACCGCAGGTGTAT GTGTATCCGCCGAGCCGCGATGAACTGACCAAAAACCAGGTGAGCCTGACCTGC CTGGTGAAAGGCTTTTATCCGAGCGATATTGCGGTGGAATGGGAAAGCAACGGC CAGCCGGAAAACAACTATAAAACCACCCCGCCGGTGCTGGATAGCGATGGCAGC TTTGCGCTGGTGAGCAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAAC GTGTTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAA AGCCTGAGCCTGAGCCCGGGC (SEQ ID NO: 47). In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a nucleic acid sequence encoding a signal peptide (e.g., SEQ ID NO: 25) immediately followed by SEQ ID NO: 47. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 47 can be determined from the amino acid sequence of SEQ ID NO: 38 as identified herein by a person of skill in the art. In some embodiments, the nucleic acid sequence encoding anti-PD-1 VH portion of an anti-PD- 1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 86: CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTC AAGATCTCCTGCAAGGCTTCTGGTTATACCTTCACAGACTATTCAATGCACTGGG TGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAAAATTGAGA CTGGTAACCCAACATATGCAGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGA AACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACAC GGCTACATATTTCTGTGCGAGAGACTACTATGGTTATTATTACTATGCTATGGACT ACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 86). In some embodiments, the nucleic acid sequence encoding anti-PD-1 VL portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 87: CAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCTAGGGGAACGGG TCACCATGACCTGCACTGTTAGCTCAAGTATAAGTTCCAGTTACTTGCACTGGTA CCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTATAGCACATCCAACCTG ActiveUS 209758784 54Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 GCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTC TCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCACCAGTA TCATCGTTCCCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 87).

[0149] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 48. GAAGTGCAGCTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTG CGCCTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCCATTATGGCATGAGCTGGG TGCGCCAGACCCCGGATAAACGCCTGGAACTGGTGGCGACCATTGATAACAACG GCGGCAACACCTATTATCCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGCG ATAACGCGAAAAACACCCTGTATCTGCAGATGAGCAGCCTGAAAAGCGAAGATA CCGCGATGTATTATTGCGCGCGCGATGCGTATTATACCTATGATTATTGGTATTTT GATGTGTGGGGCGCGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGC GGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGATATTCAGATGACCCAGACCACC AGCAGCCTGAGCGCGAGCCTGGGCGATCGCGTGACCATTAGCTGCAGCGCGAGC CAGGGCATTAGCAACTATCTGAACTGGTATCAGCAGAAACCGGATGGCACCGTG AAACTGCTGATTTATTATACCAGCAGCCTGCATAGCGGCGTGCCGAGCCGCTTTA GCGGCAGCGGCAGCGGCACCGATTATAGCCTGACCATTAGCAACCTGGAACCGG AAGATATTGCGACCTATTATTGCCAGCAGTATCATAAACTGCCGTGGACCTTTGG CGGCGGCACCAAACTGGAAATTAAACTGGAACCGAAAAGCTGCGATAAAACCCA TACCTGCCCGCCGTGCCCGGCGCCGGAATTTCAGGGCGGCCCGAGCGTGTTTCTG TTTCCGCCGAAACCGAAAGATACCCTGTATATTACCCGCGAACCGGAAGTGACCT GCGTGGTGGTGGATGTGAGCCATGAAGATCCGGAAGTGAAATTTAACTGGTATG TGGATGGCGTGGAAGTGCATAACGCGAAAACCAAACCGCGCGAAGAACAGTAT AACAGCACCTATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTG AACGGCAAAGAATATAAATGCCAGGTGAGCAACAAAGCGCTGCCGGCGCCGATT GAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACCGCAGGTGTATGT GTATCCGCCGAGCCGCGATGAACTGACCAAAAACCAGGTGAGCCTGACCTGCCT GGTGAAAGGCTTTTATCCGAGCGATATTGCGGTGGAATGGGAAAGCAACGGCCA GCCGGAAAACAACTATAAAACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTT ActiveUS 209758784 55Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 TGCGCTGGTGAGCAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGT GTTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAG CCTGAGCCTGAGCCCGGGC (SEQ ID NO: 48). In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a nucleic acid sequence encoding a signal peptide (e.g., SEQ ID NO: 25) immediately followed by SEQ ID NO: 48. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 48 can be determined from the amino acid sequence of SEQ ID NO: 39 as identified herein by a person of skill in the art. In some embodiments, the nucleic acid sequence encoding anti-PD-1 VH portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 88: GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTG AGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTCACTATGGCATGTCTTGGGT TCGCCAGACTCCAGACAAGAGGCTGGAGTTGGTCGCAACCATTGATAATAATGG TGGTAACACCTATTATCCAGACAGTGTGAAGGGCCGATTCACCATCTCCAGAGAC AATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACACA GCCATGTATTACTGTGCAAGAGACGCCTACTATACTTACGACTACTGGTACTTCG ATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 88). In some embodiments, the nucleic acid sequence encoding anti-PD-1 VL portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 89: GATATCCAGATGACACAGACTACATCCTCCCTGTCTGCCTCTCTGGGAGACAGAG TCACCATCAGTTGCAGTGCGAGTCAGGGCATTAGTAATTATTTAAACTGGTATCA GCAGAAACCAGATGGAACTGTGAAACTCCTGATCTATTACACATCAAGTTTACAT TCAGGAGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCA CCATCAGCAACCTAGAACCTGAAGATATTGCCACTTACTATTGTCAACAGTATCA TAAGCTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO: 89).

[0150] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 49. ActiveUS 209758784 56Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 GAAGTGAAACTGGTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCT GAAACTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCAGCTATACCATGAGCTG GGTGCGCCAGACCCCGGAAAAACGCCTGGAATGGGTGGCGTATATTAGCTATGG CGGCGGCGATACGTATTATCCGGATACCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACGCGAAAAACACCCTGTATCTGCAGATGAGCAGCCTGAAAAGCGAAGAT ACCGCGATGTATTATTGCGCGCGCCAGAAAGTGGATGGCTATTTTGTGGCGATGG ATTATTGGAGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCG GCGGCGGCGGCAGCGGCGGCGGCGGCAGCGTGATTGTGCTGACCCAGAGCCCGG CGAGCCTGGCGGTGAGCCTGGGCCAGCGCGCGACCATTAGCTGCCGCGCGAGCG AACGCGTGGATGATTATGGCATTAGCTTTATGAACTGGTTTCAGCAGAAACCGGG CCAGCCGCCGAAACTGCTGATTTATGCGGCGAGCAACCAGGGCAGCGGCGTGCC GGCGCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTAGCCTGAACATTCATCCG ATGGAAGAAGATGATACCGCGATGTATTTTTGCCAGCAGAACAAAGAAGTGCCG TGGACCTTTGGCGGCGGCACCAAACTGGAAATTAAACTGGAACCGAAAAGCTGC GATAAAACCCATACCTGCCCGCCGTGCCCGGCGCCGGAATTTCAGGGCGGCCCG AGCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGTATATTACCCGCGAAC CGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATGAAGATCCGGAAGTGAAAT TTAACTGGTATGTGGATGGCGTGGAAGTGCATAACGCGAAAACCAAACCGCGCG AAGAACAGTATAACAGCACCTATCGCGTGGTGAGCGTGCTGACCGTGCTGCATC AGGATTGGCTGAACGGCAAAGAATATAAATGCCAGGTGAGCAACAAAGCGCTGC CGGCGCCGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACCG CAGGTGTATGTGTATCCGCCGAGCCGCGATGAACTGACCAAAAACCAGGTGAGC CTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATATTGCGGTGGAATGGGAA AGCAACGGCCAGCCGGAAAACAACTATAAAACCACCCCGCCGGTGCTGGATAGC GATGGCAGCTTTGCGCTGGTGAGCAAACTGACCGTGGATAAAAGCCGCTGGCAG CAGGGCAACGTGTTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTAT ACCCAGAAAAGCCTGAGCCTGAGCCCGGGC (SEQ ID NO: 49). In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti- PD-1 x anti-IL-25 bispecific antibody comprises a nucleic acid sequence encoding a signal peptide (e.g., SEQ ID NO: 25) immediately followed by SEQ ID NO: 49. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 49 can be ActiveUS 209758784 57Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 determined from the amino acid sequence of SEQ ID NO: 40 as identified herein by a person of skill in the art. In some embodiments, the nucleic acid sequence encoding anti-PD-1 VH portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 90: GAAGTGAAGCTGGTGGAGTCTGGGGGAGGTTTAGTGCAGCCTGGAGGGTCCCTG AAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATACCATGTCTTGGGT TCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTTATGGTGG AGGTGACACGTACTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGA CAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAAGTCTGAGGACAC GGCCATGTATTACTGTGCCAGACAGAAGGTCGATGGTTACTTCGTTGCTATGGAC TATTGGAGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 90). In some embodiments, the nucleic acid sequence encoding anti-PD-1 VL portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 91: GTCATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGG CCACCATCTCCTGCAGAGCCAGCGAGCGTGTTGATGATTATGGCATTAGTTTTAT GAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGC ATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGAC AGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTC TGTCAGCAAAATAAGGAGGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAA ATCAAA (SEQ ID NO: 91).

[0151] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 50. GAAGTGAAACTGGTGGAAAGCGGCGGCGGCCTGGTGAAACCGGGCGGCAGCCT GAAACTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCAGCTATGGCATGAGCTG GGTGCGCCAGACCCCGGAAAAACGCCTGGAATGGGTGGCGACCATTAGCGGCGG CGGCCGCTATACCTATTATCCGGATAGCGTGAAAGGCCGCTTTACCATTAGCCGC GATAACGCGAAAAACATTCTGTATCTGCAGATGAGCAGCCTGCGCAGCGAAGAT ACCGCGCTGTATTATTGCGCGAGCCCGTATGATGGCTATTATGGCGCGATGGATT ATTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGGCGGCGGCGGCAGCGATATTGTGCTGACCCAGAGCCCGGCG ActiveUS 209758784 58Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 AGCCTGGCGGTGAGCCTGGGCCAGCGCGCGACCATTAGCTGCCGCGCGAGCGAA AGCGTGGATAACAGCGGCATTAGCTTTATGAACTGGTTTCAGCAGAAACCGGGC CAGCCGCCGAAACTGCTGATTTATGCGGCGAGCAACCAGGGCAGCGGCGTGCCG GCGCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTAGCCTGAACATTCATCCGA TGGAAGAAGATGATACCGCGATGTATTTTTGCCAGCAGAGCAAAGAAGTGCCGT GGACCTTTGGCGGCGGCACCAAACTGGAAATTAAACTGGAACCGAAAAGCTGCG ATAAAACCCATACCTGCCCGCCGTGCCCGGCGCCGGAATTTCAGGGCGGCCCGA GCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGTATATTACCCGCGAACC GGAAGTGACCTGCGTGGTGGTGGATGTGAGCCATGAAGATCCGGAAGTGAAATT TAACTGGTATGTGGATGGCGTGGAAGTGCATAACGCGAAAACCAAACCGCGCGA AGAACAGTATAACAGCACCTATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCA GGATTGGCTGAACGGCAAAGAATATAAATGCCAGGTGAGCAACAAAGCGCTGCC GGCGCCGATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACCGC AGGTGTATGTGTATCCGCCGAGCCGCGATGAACTGACCAAAAACCAGGTGAGCC TGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATATTGCGGTGGAATGGGAAA GCAACGGCCAGCCGGAAAACAACTATAAAACCACCCCGCCGGTGCTGGATAGCG ATGGCAGCTTTGCGCTGGTGAGCAAACTGACCGTGGATAAAAGCCGCTGGCAGC AGGGCAACGTGTTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATA CCCAGAAAAGCCTGAGCCTGAGCCCGGGC (SEQ ID NO: 50). In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti- IL-25 bispecific antibody comprises a nucleic acid sequence encoding a signal peptide (e.g., SEQ ID NO: 25) immediately followed by SEQ ID NO: 50. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 50 can be determined from the amino acid sequence of SEQ ID NO: 41 as identified herein by a person of skill in the art. In some embodiments, the nucleic acid sequence encoding anti-PD-1 VH portion of an anti-PD- 1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 92: GAAGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTG AAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATGGCATGTCTTGGGT TCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATCAGTGGTGGTGG TAGATACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGA CAATGCCAAGAACATCCTGTACCTGCAAATGAGTAGTCTGAGGTCTGAGGACAC ActiveUS 209758784 59Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 GGCCTTGTATTACTGTGCAAGCCCCTATGATGGTTACTACGGTGCTATGGACTAC TGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA (SEQ ID NO: 92). In some embodiments, the nucleic acid sequence encoding anti-PD-1 VL portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 93: GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGG CCACCATCTCCTGCAGAGCCAGCGAAAGTGTTGATAATTCTGGCATTAGTTTTAT GAACTGGTTCCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGC ATCCAACCAAGGATCCGGGGTCCCTGCCAGGTTTAGTGGCAGTGGGTCTGGGAC AGACTTCAGCCTCAACATCCATCCTATGGAGGAGGATGATACTGCAATGTATTTC TGTCAGCAAAGTAAGGAGGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAA ATCAAA (SEQ ID NO: 93).

[0152] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACA GC (SEQ ID NO: 25). In some embodiments, the nucleic acid sequence encoding the anti- PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) of SEQ ID NO: 76 can be determined from the amino acid sequence by a person of skill in the art.

[0153] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 51. GAAGTGCAGCTGCAGCAGAGCGGCACCGTGCTGGCGCGCCCGGGCGCGAGCGTG CGCATGAGCTGCAAAGCGAGCGGCTATAGCTTTAGCACCTATTGGATGCATTGG GTGAAACAGCGCCCGGGCCAGGGCCTGGAATGGATTGGCGGCATTTATCCGGGC AACAGCGACACCAACTATAACCAGAAATTTAAAGGCAAAGCGAAACTGACCGCG GTGACCAGCGCGAGCACCGCGTATATGGATCTGAGCAGCCTGACCAACGAAGAT AGCGCGGTGTATTATTGCACCGGCGGCTATTTTTTTGATGTGTGGGGCGCGGGCA CCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGCAGCGGC GGCGGCGGCAGCGATATTAAAATGACCCAGAGCCCGAGCAGCCTGTATGCGAGC CTGGGCGAACGCGTGACCATTACCTGCAAAGCGAGCCAGGATATTAACCGCTAT ActiveUS 209758784 60Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 CTGACCTGGTTTCAGCAGAAACCGGGCAAAAGCCCGAAAACCCTGATTTTTCGC GCGAACCGCCTGGTGACCGGCGTGCCGAGCCGCTTTATTGGCAGCGGCAGCGGC CAGGAATATAGCCTGACCATTAGCAGCCTGGAATATGAAGATATGGGCATTTATT TTTGCCTGCAGTATGATGAAAGCCCGTATACCTTTGGCGGCGGCACCAAACTGGA AATTAAACTGGAACCGAAAAGCTGCGATAAAACCCATACCTGCCCGCCGTGCCC GGCGCCGGAATTTCAGGGCGGCCCGAGCGTGTTTCTGTTTCCGCCGAAACCGAA AGATACCCTGTATATTACCCGCGAACCGGAAGTGACCTGCGTGGTGGTGGATGT GAGCCATGAAGATCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGTGGAAGT GCATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCTATCGCGT GGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAAGAATATAA ATGCCAGGTGAGCAACAAAGCGCTGCCGGCGCCGATTGAAAAAACCATTAGCAA AGCGAAAGGCCAGCCGCGCGAACCGCAGGTGTATGTGTATCCGCCGAGCCGCGA TGAACTGACCAAAAACCAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCC GAGCGATATTGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAACTATA AAACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTGCGCTGGTGAGCAAAC TGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGTGTTTAGCTGCAGCGTGA TGCATGAAGCGCTGCATAACCATTATACCCAGAAAAGCCTGAGCCTGAGCCCGG GC (SEQ ID NO: 51). In some embodiments, the nucleic acid sequence encoding the anti- PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a nucleic acid sequence encoding a signal peptide (e.g., SEQ ID NO: 25) immediately followed by SEQ ID NO: 51. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 51 can be determined from the amino acid sequence of SEQ ID NO: 42 as identified herein by a person of skill in the art. In some embodiments, the nucleic acid sequence encoding anti-PD-1 VH portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 94: GAGGTTCAGCTCCAGCAGTCTGGGACTGTGCTGGCAAGGCCTGGGGCTTCCGTG AGGATGTCCTGCAAGGCTTCTGGCTACAGCTTTTCCACCTACTGGATGCACTGGG TAAAACAGAGGCCTGGACAGGGTCTAGAATGGATTGGTGGTATTTATCCTGGAA ATAGTGACACTAACTACAACCAGAAGTTCAAGGGCAAGGCCAAACTGACTGCAG TCACTTCCGCCAGCACTGCCTACATGGACCTCAGCAGCCTGACAAATGAGGACTC TGCGGTCTATTACTGTACAGGCGGTTACTTCTTCGATGTCTGGGGCGCAGGGACC ActiveUS 209758784 61Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 ACGGTCACCGTCTCCTCA (SEQ ID NO: 94). In some embodiments, the nucleic acid sequence encoding anti-PD-1 VL portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 95: GACATCAAGATGACCCAGTCTCCATCTTCCTTGTATGCATCTCTAGGAGAGAGAG TCACTATCACTTGCAAGGCGAGTCAGGACATTAATCGATATTTAACCTGGTTCCA GCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTTTCGTGCAAACAGATTGGT AACTGGGGTCCCATCAAGGTTCATTGGCAGTGGATCTGGGCAAGAATATTCTCTC ACCATCAGCAGCCTGGAGTATGAAGACATGGGAATTTATTTTTGTCTACAGTATG ATGAGTCTCCGTACACGTTCGGAGGGGGGACCAAGTTGGAAATAAAA (SEQ ID NO: 95).

[0154] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 52. CAGATTCAGCTGCAGCAGAGCGGCCCGGAACAGGTGAAACCGGGCGCGAGCGT GAAAATTAGCTGCAAAGCGAGCGGCTATATGTTTATTGATTATTATATGAACTGG GTGAAACAGAAACCGGGCCAGGGCCTGGAATGGATTGGCTGGATTTATCCGGGC AGCGGCAGCACCAAAGATAACGAAAACTTTAAAGGCAAAGCGACCCTGACCGTG GATACCAGCAGCAGCACCGCGTATATGCAGCTGAGCAGCCTGACCAGCGAAGAT ACCGCGGTGTATTTTTGCGCGCGCTATGGCCCGCGCTTTTTTGATGTGTGGGGCG CGGGCACCACCGTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGC AGCGGCGGCGGCGGCAGCGATATTCAGATGACCCAGAGCCCGAGCAGCCTGAGC GCGAGCCTGGGCGAACGCGTGAACCTGACCTGCCGCGCGAGCCAGGAAATTAGC GGCTATCTGAGCTGGCTGCAGCAGAAACCGGATGGCACCATTAAACGCCTGATT TATGTGGCGAGCACCCTGGATAGCGGCGTGCCGGAACGCTTTAGCGGCAGCCGC AGCGGCAGCGATTATAGCCTGACCATTAGCAGCCTGGAAAGCGAAGATTTTGCG GATTATTATTGCCTGCAGTATGCGAGCTATCCGTATACCTTTGGCGGCGGCACCA AACTGGAAATTAAACTGGAACCGAAAAGCTGCGATAAAACCCATACCTGCCCGC CGTGCCCGGCGCCGGAATTTCAGGGCGGCCCGAGCGTGTTTCTGTTTCCGCCGAA ACCGAAAGATACCCTGTATATTACCCGCGAACCGGAAGTGACCTGCGTGGTGGT GGATGTGAGCCATGAAGATCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGT GGAAGTGCATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCT ActiveUS 209758784 62Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 ATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAAG AATATAAATGCCAGGTGAGCAACAAAGCGCTGCCGGCGCCGATTGAAAAAACCA TTAGCAAAGCGAAAGGCCAGCCGCGCGAACCGCAGGTGTATGTGTATCCGCCGA GCCGCGATGAACTGACCAAAAACCAGGTGAGCCTGACCTGCCTGGTGAAAGGCT TTTATCCGAGCGATATTGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACA ACTATAAAACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTGCGCTGGTGA GCAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGTGTTTAGCTGCA GCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAGCCTGAGCCTGA GCCCGGGC (SEQ ID NO: 52). In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a nucleic acid sequence encoding a signal peptide (e.g., SEQ ID NO: 25) immediately followed by SEQ ID NO: 52. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 52 can be determined from the amino acid sequence of SEQ ID NO: 43 as identified herein by a person of skill in the art. In some embodiments, the nucleic acid sequence encoding anti-PD-1 VH portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 96: CAGATCCAGCTGCAGCAGTCTGGACCTGAGCAGGTGAAGCCTGGGGCTTCAGTG AAGATATCCTGCAAGGCTTCTGGCTACATGTTCATCGACTACTATATGAACTGGG TGAAGCAGAAGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTGGAA GCGGTAGTACTAAGGACAATGAGAATTTCAAGGGCAAGGCCACATTGACTGTAG ACACATCCTCCAGCACAGCCTACATGCAGCTCAGTAGCCTGACATCTGAGGACA CTGCTGTCTATTTCTGTGCAAGATATGGGCCCAGGTTCTTCGATGTCTGGGGCGC AGGGACCACGGTCACCGTCTCCTCA (SEQ ID NO: 96). In some embodiments, the nucleic acid sequence encoding anti-PD-1 VL portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 97: GACATCCAGATGACCCAGTCTCCATCCTCCTTATCTGCCTCTCTGGGAGAAAGAG TCAATCTCACTTGTCGGGCAAGTCAGGAAATTAGTGGTTACTTAAGCTGGCTTCA GCAGAAACCAGATGGAACTATTAAACGCCTGATCTACGTCGCATCCACTTTAGAT TCTGGTGTCCCAGAAAGGTTCAGTGGCAGTAGGTCTGGGTCAGATTATTCTCTCA CCATCAGCAGCCTTGAGTCTGAGGATTTTGCAGACTATTACTGTCTACAATATGC ActiveUS 209758784 63Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 TAGTTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA (SEQ ID NO: 97).

[0155] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises SEQ ID NO: 53. GAAGTGAAACTGCTGGAAAGCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCCTG AAACTGAGCTGCGAAGTGAGCGGCTTTGATTTTAGCCGCGATTGGATGAGCTGG GTGCGCCAGGCGCCGGGCAAAGGCCTGGAATGGATTGGCCAGATTAGCCCGGAT AGCACCAGCATTAACTATAAACCGAGCCTGAAAGATAAATTTATTATTAGCCGC GATAACGCGAAAAACACCCTGTATCTGCAGCTGAGCGGCGTGCGCAGCGAAGAT ACCGCGCTGTATCATTGCGCGCGCGATACCAGCGGCTATCCGGATTATTGGGGCC AGGGCACCAGCCTGACCGTGAGCAGCGGCGGCGGCGGCAGCGGCGGCGGCGGC AGCGGCGGCGGCGGCAGCGATATTCTGCTGACCCAGAGCCCGAGCAGCATGAGC GTGAGCCTGGGCGATACCGTGAGCATTACCTGCCATGCGAGCCAGGATATTAGC CGCAACATTGGCTGGCTGCGCCAGAAACCGGGCAAAAGCTTTAAAGGCCTGATT TATCATGGCACCAACCTGGAAGATGGCGTGCCGAGCCGCTTTAGCGGCAGCGGC AGCGGCGCGGATTATAGCCTGACCATTAGCAGCCTGGAAAGCGAAGATTTTGCG GATTATTATTGCGTGCAGTATGCGCAGTTTCCGTATACCTTTGGCGGCGGCACCA AACTGGAAATTAAACTGGAACCGAAAAGCTGCGATAAAACCCATACCTGCCCGC CGTGCCCGGCGCCGGAATTTCAGGGCGGCCCGAGCGTGTTTCTGTTTCCGCCGAA ACCGAAAGATACCCTGTATATTACCCGCGAACCGGAAGTGACCTGCGTGGTGGT GGATGTGAGCCATGAAGATCCGGAAGTGAAATTTAACTGGTATGTGGATGGCGT GGAAGTGCATAACGCGAAAACCAAACCGCGCGAAGAACAGTATAACAGCACCT ATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAAG AATATAAATGCCAGGTGAGCAACAAAGCGCTGCCGGCGCCGATTGAAAAAACCA TTAGCAAAGCGAAAGGCCAGCCGCGCGAACCGCAGGTGTATGTGTATCCGCCGA GCCGCGATGAACTGACCAAAAACCAGGTGAGCCTGACCTGCCTGGTGAAAGGCT TTTATCCGAGCGATATTGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACA ACTATAAAACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTGCGCTGGTGA GCAAACTGACCGTGGATAAAAGCCGCTGGCAGCAGGGCAACGTGTTTAGCTGCA GCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAGCCTGAGCCTGA ActiveUS 209758784 64Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 GCCCGGGC (SEQ ID NO: 53). In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody comprises a nucleic acid sequence encoding a signal peptide (SEQ ID NO: 25) immediately followed by SEQ ID NO: 53. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO: 53 can be determined from the amino acid sequence of SEQ ID NO: 44 as identified herein by a person of skill in the art. In some embodiments, the nucleic acid sequence encoding anti-PD-1 VH portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 98: GAGGTGAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTG AAACTCTCCTGTGAAGTCTCAGGATTCGATTTTAGCAGAGACTGGATGAGTTGGG TCCGGCAGGCTCCAGGGAAAGGGCTAGAGTGGATTGGACAAATTTCTCCAGATA GCACTTCGATAAACTATAAGCCATCTCTAAAGGATAAATTCATCATCTCCAGAGA CAACGCCAAAAATACGCTGTACCTGCAATTGAGCGGAGTGAGATCTGAGGACAC AGCCCTTTATCACTGTGCAAGAGACACCTCGGGCTACCCCGACTACTGGGGCCAA GGCACCAGTCTCACAGTCTCCTCA (SEQ ID NO: 98). In some embodiments, the nucleic acid sequence encoding anti-PD-1 VL portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 99: GACATCCTGTTGACCCAATCTCCATCCTCCATGTCTGTTTCTCTGGGAGACACAGT CAGCATAACGTGTCATGCAAGTCAGGACATTAGCAGGAATATAGGGTGGTTGCG GCAGAAACCAGGGAAATCATTTAAGGGCCTGATCTATCATGGAACCAATTTGGA AGATGGAGTTCCATCAAGGTTCAGCGGCAGTGGATCTGGAGCAGATTATTCTCTC ACCATCAGCAGCCTGGAATCTGAAGATTTTGCAGACTATTACTGTGTGCAGTATG CTCAGTTTCCGTACACGTTCGGAGGGGGGACCAAACTGGAAATAAAA (SEQ ID NO: 99).

[0156] In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a signal peptide comprising SEQ ID NO: 25. ATGGGCTGGAGCTGCATTATCCTGTTCCTGGTGGCCACAGCCACCGGCGTGCACA GC (SEQ ID NO: 25). In some embodiments, the nucleic acid sequence encoding the anti- PD-1 portion (scFv-Fc) of an anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) of SEQ ID NO: 79 can be determined from the amino acid sequence by a person of ActiveUS 209758784 65Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 skill in the art. Nucleic acid sequences encoding the respective CDRs, linker portions, VH portion, VL portion, Fc portion and knob / hole mutations in SEQ ID NO:79 can be determined from the amino acid sequence of SEQ ID NO: 79 as identified herein by a person of skill in the art. In some embodiments, the nucleic acid sequence encoding anti-PD-1 VH portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO 100: GAAGTGAAGCTGGTGGAGTCTGGGGGAGGTTTAGTGCAGCCTGGAGGGTCCCTG AAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTATACCATGTCTTGGGT TCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTTATGGTGG AGGTGACACCTACTATTCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGA CAATGCCAAGAACACCCTGTACCTACAAATGAGCAGTCTGAAGTCTGAGGACAC GGCCATGTATTACTGTGCAAGACAGAAGGTCGATGGTTACTACGTTGGTATGGAC TACTGGGGTCAAGGATCCTCAGTCACCGTCTCCTCA (SEQ ID NO: 100). In some embodiments, the nucleic acid sequence encoding anti-PD-1 VL portion of an anti-PD-1 x anti-IL-25 bispecific antibody (without a signal peptide) comprises SEQ ID NO: 101: AACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGG GTCACCTTGAGCTGCAAGGCCAGCGAGAATGTGGGTACTAATGTATCCTGGTATC AACAGAAACCAGAGCAGTCTCCTAGACTGCTGATATATGGGGCTTCCAACCGGT ACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGTAACAGATTTCACTCT GACCATCAGCAGTGTGCAGGCTGAGGACCTTGTAGATTATCACTGTGGACAGAG TTACAGCTCTCCGCTCACGTTCGGTTCTGGGACCAAGCTGGAGCTGAAA (SEQ ID NO: 101).

[0157] In some embodiments, the nucleic acid sequence encoding a signal peptide of the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 25. In some embodiments, the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 26. In some embodiments, the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti- PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 45. In some embodiments, the nucleic acid sequence encoding an ActiveUS 209758784 66Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 46. In some embodiments, the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 47. In some embodiments, the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 48. In some embodiments, the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 49. In some embodiments, the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 50. In some embodiments, the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 51. In some embodiments, the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 52. In some embodiments, the nucleic acid sequence encoding an anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NO: 53. In some embodiments, the nucleic acid sequence encoding the VH of an anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, 98, or 100. In some embodiments, the nucleic acid sequence encoding the VL of an anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody (without the signal peptide) comprises an nucleic acid sequence 80, 81, ActiveUS 209758784 67Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, 99 or 101. In some embodiments, the nucleic acid sequence encodes CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 portion (scFv-Fc) of the anti-PD-1 x anti-IL-25 bispecific antibody wherein the CDR sequences are indicated above (in bold and underline) or in Figure 10 or Tables 1 and 3. In some embodiments, the nucleic acid sequence encoding framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise an nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 26. In some embodiments, the nucleic acid sequence encoding framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 % identical to the FRs of SEQ ID NOs: 45-53. In some embodiments, the nucleic acid sequence encoding framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprise a nucleic acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 % identical to the FRs of Tables 2 and 4.

[0158] Although the sequence above is depicted with a nucleic acid sequence encoding a linker “GGAGGCGGAGGATCTGGTGGCGGAGGAAGTGGCGGAGGCGGTTCT” (SEQ ID NO: 27) a person of skill in the art recognizes that numerous other flexible linkers known in the art can be used, including those enriched in small or hydrophilic amino acids. In some embodiments, the linker between the VH and VL domains comprises a glycine-serine linker. In some embodiments, any combination of glycine and serine residues can be used. In some embodiments, the glycine-serine linker comprises a nucleic acid sequence encoding a linker that is between about 2 and 25 amino acids in length. In some embodiments, the nucleic acid sequence encodes a glycine-serine linker that is about 15 amino acids in length. Additional Amino Acid and Nucleic Acid Sequences of An Anti-PD-1 Portion of Anti-PD-1 x Anti-IL-25 Bispecific Antibodies

[0159] In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises the PD-1 binding portion of clone C2C of US2020 / 0002420, the contents of which is hereby incorporated by reference in its entirety. ActiveUS 209758784 68Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0160] In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a single-chain variable fragment (scFv) with a fragment crystallizable (Fc) region (i.e., scFv-Fc antibody) comprises a signal peptide, a variable heavy chain domain, a linker, a variable light chain domain, and an Fc portion. In some embodiments, the amino acid sequence of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises at least a portion of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab, Camrelizumab, Sintilimab, Penpulimab, clone C2C of US2020 / 0002420, or any other anti- PD-1 antibody known in the art. In some embodiments, the amino acid sequence of the anti- PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises at least a variable heavy and variable light chain portions of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab, Camrelizumab, Sintilimab, Penpulimab, clone C2C of US2020 / 0002420, or any other anti- PD-1 antibody known in the art. In some embodiments, the amino acid sequence of the anti- PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises at least the CDRs of the variable heavy chain and the CDRs of the variable light chain portions of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab, Camrelizumab, Sintilimab, Penpulimab, clone C2C of US2020 / 0002420, or any other anti-PD-1 antibody known in the art In some embodiments, the nucleic acid sequence the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody codes for an amino acid sequence that comprises at least a portion of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab, Camrelizumab, Sintilimab, Penpulimab, clone C2C of US2020 / 0002420, or any other anti-PD-1 antibody known in the art. In some embodiments, the nucleic acid sequence of the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody codes for an amino acid sequence that comprises at least a variable heavy and variable light chain portions of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab, Camrelizumab, Sintilimab, Penpulimab, clone C2C of US2020 / 0002420, or any other anti-PD-1 antibody known in the art. In some embodiments, the nucleic acid sequence encoding the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody codes for an amino acid sequence that comprises at least the CDRs of the variable heavy chain and the CDRs of the variable light chain portions of the amino acid sequence of Nivolumab, Pembrolizumab, Cemiplimab, ActiveUS 209758784 69Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 Retifanlimab, Dostarlimab, Zimberelimab, Tiselelizumab, Camrelizumab, Sintilimab, Penpulimab, clone C2C of US2020 / 0002420, or any other anti-PD-1 antibody known in the art. The sequences of anti-PD-1 antibodies are described in the art and incorporated herein by reference as follows: Pembrolizumab (see USPN 8,168,757, USPN 8,354,509, USPN 8,900,587, the contents of each of which is hereby incorporated by reference in its entirety), Cemiplimab (see USPN 9,987,500 the contents of which is hereby incorporated by reference in its entirety), Retifanlimab (see US2019 / 0127467 the contents of which is hereby incorporated by reference in its entirety), Dostarlimab (see WO / 2021 / 058711 the contents of which is hereby incorporated by reference in its entirety), Zimberelimab (see CN106432494 the contents of which is hereby incorporated by reference in its entirety), Tiselelizumab (see USPN 8,735,553 the contents of which is hereby incorporated by reference in its entirety), Camrelizumab (see US2019 / 0309069 the contents of which is hereby incorporated by reference in its entirety), Sintilimab (see USPN 10,316,089 the contents of which is hereby incorporated by reference in its entirety), Penpulimab (see US2019 / 0321466 the contents of which is hereby incorporated by reference in its entirety), clone C2C (see US2020 / 0002420 the contents of which is hereby incorporated by reference in its entirety). The CDRs of certain anti-PD-1 antibodies are described in Jeong TJ, Lee HT, Gu N, Jang YJ, Choi SB, Park UB, Lee SH, Heo YS, The High-Resolution Structure Reveals Remarkable Similarity in PD-1 Binding of Cemiplimab and ostarlimab, the FDA-Approved Antibodies for Cancer Immunotherapy. Biomedicines, 2022 Dec 6;10(12):3154, the contents of which is hereby incorporated by reference in its entirety.

[0161] In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain with hypervariable regions HCDR1, HCDR2 and HCDR3. HCDR1 comprising the amino acid sequence SEQ ID NO: 28 (TYYIS), HCDR2 comprising the amino acid sequence SEQ ID NO: 29 (YINMGSGGTNYNEKFKG), and HCDR3 comprising the amino acid sequence SEQ ID NO: 30 (IGYFDY); or HCDR equivalents thereof. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL- 25 bispecific antibody comprises a VL domain which comprises hypervariable regions LCDR1, LCDR2, and LCDR3. LCDR1 comprising the amino acid sequence SEQ ID NO: 31 (RSSQSLLDSDGATYLY), LCDR2 comprising the amino acid sequence SEQ ID NO: 32 (LVSTLGS), and LCDR3 comprising the amino acid sequence SEQ ID NO: 33 (MQLTHWPYT), or LCDR equivalents thereof. ActiveUS 209758784 70Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0162] In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 34 (QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSG GTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQGTMVT VSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 35 (DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGATYLYWFQQRPGQSPRRLIYLVST LGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLEIK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 54 (EVKLLESGGGLVQPGGSLKLSCAASGFDFSRYWMSWVRQAPGKGLEWIGEINPDSS TIKYTPSLKDKFIISRDNAKNTLCLQLSKVRSEDSALYYCARMGYRLFDSWGQGTTL TVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 55 (NIVMTQATPSVPVTPGESVSISCRSSKSLLHSDGDTYLYWFLQRPGQSPQLLIYRMSN LVSGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPYTFGGGTKLEIK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 56 (EVQLQQSGTVLARPGASVKMSCKASGNTFTSYWMHWVKQRPGQGLEWIGAIYPG YSDTRYNQKFKGKATLTAVTSTSTAYMELSSLTNEDSAVYYCTKFIATVGGYFDVW GAGTTVTVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 57 (QIVLTQSPAIMSSSLGERVTMTCTASSSVSSSYLHWYQQKPGSSPKLWIYSTSNLASG VPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPPIFTFGSGTKLEIK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 58 (QIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKGLKWMGWIKIET GNPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARDYYGYYYYAMDY WGQGTSVTVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL- 25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 59 (QIVLTQSPAIMSASLGERVTMTCTVSSSISSSYLHWYQQKPGSSPKLWIYSTSNLASG VPARFSGSGSGTSYSLTISSMEAEDAATYYCHQYHRSPLTFGAGTKLELK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody ActiveUS 209758784 71Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 comprises a VH domain comprising SEQ ID NO: 60 (EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYGMSWVRQTPDKRLELVATIDNNGG NTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARDAYYTYDYWYFDV WGAGTTVTVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL- 25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 61 (DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYYTSSLHSG VPSRFSGSGSGTDYSLTISNLEPEDIATYYCQQYHKLPWTFGGGTKLEIK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 62 (EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVAYISYGGG DTYYPDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQKVDGYFVAMDY WSQGTSVTVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL- 25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 63 (VIVLTQSPASLAVSLGQRATISCRASERVDDYGISFMNWFQQKPGQPPKLLIYAASN QGSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQNKEVPWTFGGGTKLEIK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 64 (EVKLVESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPEKRLEWVATISGGGR YTYYPDSVKGRFTISRDNAKNILYLQMSSLRSEDTALYYCASPYDGYYGAMDYWG QGTSVTVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 65 (DIVLTQSPASLAVSLGQRATISCRASESVDNSGISFMNWFQQKPGQPPKLLIYAASNQ GSGVPARFSGSGSGTDFSLNIHPMEEDDTAMYFCQQSKEVPWTFGGGTKLEIK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 66 (EVQLQQSGTVLARPGASVRMSCKASGYSFSTYWMHWVKQRPGQGLEWIGGIYPG NSDTNYNQKFKGKAKLTAVTSASTAYMDLSSLTNEDSAVYYCTGGYFFDVWGAGT TVTVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 67 (DIKMTQSPSSLYASLGERVTITCKASQDINRYLTWFQQKPGKSPKTLIFRANRLVTG VPSRFIGSGSGQEYSLTISSLEYEDMGIYFCLQYDESPYTFGGGTKLEIK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 68 ActiveUS 209758784 72Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 (QIQLQQSGPEQVKPGASVKISCKASGYMFIDYYMNWVKQKPGQGLEWIGWIYPGS GSTKDNENFKGKATLTVDTSSSTAYMQLSSLTSEDTAVYFCARYGPRFFDVWGAGT TVTVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 69 (DIQMTQSPSSLSASLGERVNLTCRASQEISGYLSWLQQKPDGTIKRLIYVASTLDSGV PERFSGSRSGSDYSLTISSLESEDFADYYCLQYASYPYTFGGGTKLEIK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 70 (EVKLLESGGGLVQPGGSLKLSCEVSGFDFSRDWMSWVRQAPGKGLEWIGQISPDST SINYKPSLKDKFIISRDNAKNTLYLQLSGVRSEDTALYHCARDTSGYPDYWGQGTSL TVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 71 (DILLTQSPSSMSVSLGDTVSITCHASQDISRNIGWLRQKPGKSFKGLIYHGTNLEDGV PSRFSGSGSGADYSLTISSLESEDFADYYCVQYAQFPYTFGGGTKLEIK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 77 (EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYGMSWVRQAPGKGLEWVATISGGG RYTYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCASPYDGYYGAMDYW GQGTSVTVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 78 (EIVLTQSPATLSLSPGERATLSCRASESVDNSGISFMNWYQQKPGQAPRLLIYAASNQ GSGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSKEVPWTFGGGTKLEIKR). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 80 (EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYTMSWVRQTPEKRLEWVAYISYGGG DTYYSDTVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARQKVDGYYVGMDY WGQGSSVTVSS). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL- 25 bispecific antibody comprises a VL domain comprising SEQ ID NO: 81 (NIVMTQSPKSMSMSVGERVTLSCKASENVGTNVSWYQQKPEQSPRLLIYGASNRYT GVPDRFTGSGSVTDFTLTISSVQAEDLVDYHCGQSYSSPLTFGSGTKLELK). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 34 and comprises a VL domain comprising SEQ ID NO: 35. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 ActiveUS 209758784 73Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 bispecific antibody comprises a VH domain comprising SEQ ID NO: 54 and comprises a VL domain comprising SEQ ID NO: 55. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 56 and comprises a VL domain comprising SEQ ID NO: 57. In some embodiments, the anti- PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 58 and comprises a VL domain comprising SEQ ID NO: 59. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 60 and comprises a VL domain comprising SEQ ID NO: 61. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 62 and comprises a VL domain comprising SEQ ID NO: 63. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 64 and comprises a VL domain comprising SEQ ID NO: 65. In some embodiments, the anti- PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 66 and comprises a VL domain comprising SEQ ID NO: 67. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 68 and comprises a VL domain comprising SEQ ID NO: 69. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 70 and comprises a VL domain comprising SEQ ID NO: 71. In some embodiments, the anti-PD-1 portion of the anti- PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising SEQ ID NO: 77 and comprises a VL domain comprising SEQ ID NO: 78. In some embodiments, the anti- PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to any of SEQ ID NO: 34, 54, 56, 58, 60, 62, 64, 66, 68, 70, or 73. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VH domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 34 and comprising HCDR1 comprising the amino acid sequence SEQ ID NO: 28 (TYYIS), HCDR2 comprising the amino acid sequence SEQ ID NO: 29 (YINMGSGGTNYNEKFKG), and HCDR3 comprising the amino acid sequence SEQ ID NO: 30 (IGYFDY). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL- 25 bispecific antibody comprises a VH domain comprising an amino acid sequence having ActiveUS 209758784 74Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 54, 56, 58, 60, 62, 64, 66, 68, 70 or 73 and comprising HCDR1, HCDR2, and HCDR3 corresponding to the sequences in Figure 10. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 35, 55, 57, 59, 61, 63, 65, 67, 69, 71, or 78. In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 35 and comprising LCDR1 comprising the amino acid sequence SEQ ID NO: 31 (RSSQSLLDSDGATYLY), LCDR2 comprising the amino acid sequence SEQ ID NO: 32 (LVSTLGS), and LCDR3 comprising the amino acid sequence SEQ ID NO: 33 (MQLTHWPYT). In some embodiments, the anti-PD-1 portion of the anti-PD-1 x anti-IL-25 bispecific antibody comprises a VL domain comprising an amino acid sequence having 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identity to SEQ ID NO: 55, 57, 59, 61, 63, 65, 67, 69, 71 or 78 and comprising LCDR1, LCDR2, and LCDR3 corresponding to the sequences in Figure 10. Amino Acid Sequences of Anti-PD-1 x Anti-IL-25 Bispecific Antibodies

[0163] The anti-PD-1 x anti-IL-25 bispecific antibody comprising SEQ ID NO: 1 for the anti-IL-25 portion and SEQ ID NO: 24 for the anti-PD-1 portion is represented by antibody “CEPO1.25” in embodiments described and depicted in this disclosure.

[0164] In some embodiments, the anti-PD-1 x anti-IL-25 bispecific antibody comprises SEQ ID NO: 1 for the anti-IL-25 portion and any of SEQ ID NOs: 36-44 or SEQ ID NO: 76 for the anti-PD-1 portion. Monoclonal Anti-PD-1 Antibodies

[0165] Described herein are monoclonal antibodies or antigen binding fragment thereof comprising an antigen binding site against PD-1. In some embodiments, the antibodies or antigen binding fragment thereof can be used for cancer immunotherapy and / or in combination therapies with anti-IL25 antibodies.

[0166] Described herein are amino acid sequences and nucleic acid sequences of anti-PD- 1monoclonal antibodies or antigen binding fragment thereof. The monoclonal antibodies or antigen binding fragment thereof contemplated herein can comprise an antigen binding site against PD-1 comprising an amino acid sequence of the anti-PD-1 portions described herein. ActiveUS 209758784 75Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 In some embodiments, the amino acid sequences of the anti-PD-1 portion is encoded by the nucleic acid sequences disclosed herein.

[0167] In some embodiments, the anti-PD1 antibody comprises two polypeptide heavy chains each comprising a variable heavy chain (VH) domain, CH1 domain, a hinge domain, a Fc domain (CH2-CH3) and two polypeptide light chains comprising a variable light chain (VL) domain and a CL domain. In some embodiments, the first and second chains are linked by one or more covalent disulfide bonds and the third and fourth chains are linked by one or more covalent disulfide bonds. In some embodiments, the first and third chains are linked by one or more disulfide bonds.

[0168] In some embodiments, the VH domain comprises the VH domain sequence of SEQ ID NO: 36-44 or SEQ ID NO: 76, 79 and the VL domain comprises the VL domain sequence of SEQ ID NO: 36-44 or SEQ ID NO: 76, 79. In some embodiments, the Fc domain comprises the Fc domain sequence of SEQ ID NO: 36-44. In some embodiments, the anti-PD-1 antibody heavy chain comprises a VH domain with an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VH domain sequence of SEQ ID NO: 36-44 or SEQ ID NO: 76, 79. In some embodiments, the anti-PD-1 antibody light chain comprises a VL domain with amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the VL domain sequence of SEQ ID NO: 36-44 or SEQ ID NO: 76, 79. In some embodiments, the anti-PD-1 antibody comprises an amino acid sequence comprising the CDRs of the variable heavy chain domain and the CDRs of the variable light chain domain of the anti-PD-1 antibody, wherein the CDR sequences are indicated in Figure 10 for in SEQ ID NOs: 36-44 or SEQ ID NO: 76, 79. In some embodiments, the framework regions (FRs) of the variable heavy chain domain and the FRs of the variable light chain domain of the anti-PD-1 antibody comprise an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99 % identical to the FRs of SEQ ID NO: 36-44 or SEQ ID NO: 76, 79.

[0169] In some embodiments, the amino acid sequence of the anti-PD-1 antibody heavy and light chain comprises SEQ ID NO: 1 immediately followed by the sequences of the heavy or light chains. In some embodiments, the signal peptide is cleaved during post- translational modifications that occur in vitro or in vivo.

[0170] In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising a first and second chain that associate together, each chain comprising a variable heavy chain (VH) domain of SEQ ID NO: 36-44 or SEQ ID NO: 76, 79, a linker (e.g., ActiveUS 209758784 76Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 GGGGSGGGGSGGGGS (SEQ ID NO: 2)), a variable light chain (VL) domain of SEQ ID NO: 36-44 or SEQ ID NO: 76, 79, an Fc domain of SEQ ID NO: 36-44 or SEQ ID NO: 76, 79. In some embodiments the monoclonal antibody is a scFv-Fc antibody comprising SEQ ID NO: 36-44 or SEQ ID NO: 76, 79.

[0171] In some embodiments, the amino acid sequence of the anti-PD-1 (scFv-Fc) antibody comprises a signal peptide comprising SEQ ID NO: 23. MGWSCIILFLVATATGVHS (SEQ ID NO: 23). In some embodiments, the amino acid sequence of the anti-PD-1 (scFv-Fc) antibody (without the signal peptide) comprises SEQ ID NO: 36-44 or SEQ ID NO: 76, 79. In some embodiments, the amino acid sequence of the anti-PD-1 (scFv-Fc) antibody comprises SEQ ID NO: 1 immediately followed by SEQ ID NO: 36-44 or SEQ ID NO: 76, 79. In some embodiments, the signal peptide is cleaved during post-translational modifications that occur in vitro or in vivo.

[0172] A person skilled in the art can identify the nucleic acid sequences encoding the features identified in the corresponding amino acid sequences (e.g., CDRs, variable heavy chain and variable light chain domains, Fc portion) by translating the nucleic acid sequences into amino acid sequences. In some embodiments, the nucleic acid sequence encoding the VH domain, VL domain, and / or Fc portion of an anti-PD-1 antibody comprises the VH, VL, and / or Fc encoding portions of SEQ ID NO:36-44 or SEQ ID NO: 76, 79.

[0173] In some embodiments, an anti-PD-1 antibody, or antigen binding fragment thereof, as disclosed herein, is used to prevent or treat cancer in a subject. In some embodiments, the cancer is selected from colorectal cancer, lung cancer, bladder cancer, breast cancer, cervical cancer, kidney cancer, leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, prostate cancer, skin cancer (e.g., melanoma), head and neck cancer, endometrial cancer, colon cancer, rectal cancer, liver cancer, thyroids cancer, esophageal cancer, renal cell cancer, and a combination thereof. In some embodiments, an anti-CD45 antibody, or antigen binding fragment thereof, as disclosed herein, is used to prevent or treat disease caused by any solid tumor that is not able to repair errors in its DNA that occur when the DNA is copied.

[0174] In some embodiments, the present invention relates to anti-IL25 antibodies combined with PD-1 antibodies (e.g. antibodies with variable regions or CDRs of SEQ ID NOs: 36-44 or SEQ ID NO: 76, 79) for cancer treatment and to prevent immune-related adverse events (irAEs) associated with immune checkpoint inhibitors treatments without interfering with the anti-tumor effects. In certain aspects, the present disclosure provides a ActiveUS 209758784 77Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 method for treating cancer in a subject in need thereof comprising: administering to the subject a therapeutically effective amount of an anti-IL-25 antibody or antigen binding fragment thereof (e.g. antibodies / fragments thereof with variable regions or CDRs of SEQ ID NO: 1 or any known anti-IL25 antibody) and administering to the subject a therapeutically effective amount of a PD-1 antibody, or antigen binding fragment thereof (e.g. antibodies / fragments thereof with variable regions or CDRs of SEQ ID NOs: 36-44 or SEQ ID NO: 76, 79).

[0175] In some embodiments, the anti-IL-25 antibody or antigen binding fragment thereof, comprises: a first arm comprising a first variable heavy chain domain and a first variable light chain domain of SEQ ID NO: 1 or of a known IL-25 antibody in the art; and a second arm comprising a second variable heavy chain domain and a second variable light chain domain, of SEQ ID NO: 1 or of a known IL-25 antibody in the art; wherein the first and second arms each further comprise a fragment, crystallizable (Fc) domain. In some embodiments, the first and second arms each further comprise a CH1 domain, a hinge domain, and a CL domain. In some embodiments, the portion of IL-25 bound by the first arm and second arm is the same.

[0176] In some embodiments, the anti-PD1 antibody or antigen binding fragment thereof, comprises: a first arm comprising a first variable heavy chain domain and a first variable light chain domain of SEQ ID NO: 36-44 or SEQ ID NO: 76, 79; and a second arm comprising a second variable heavy chain domain and a second variable light chain domain, of SEQ ID NO: 36-44 or SEQ ID NO: 76, 79; wherein the first and second arms each further comprise a fragment, crystallizable (Fc) domain. In some embodiments, the first and second arms each further comprise a CH1 domain, a hinge domain, and a CL domain. In some embodiments, the portion of PD-1 bound by the first arm and second arm is the same.

[0177] In some embodiments, the first variable heavy chain domain of the first arm is encoded by a first polypeptide chain; the first variable light chain domain of the first arm is encoded by a second polypeptide chain; the second variable heavy chain domain of the second arm is encoded by a third polypeptide chain; the second variable light chain domain of the second arm is encoded by a fourth polypeptide chain; and the first variable heavy chain domain and first variable light chain domain form a first IL-25 binding site and wherein the second variable heavy chain domain and second variable light chain domain form a second IL-25 binding site. In some embodiments, the first and second IL-25 binding sites are the same. ActiveUS 209758784 78Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0178] In some embodiments, the first variable heavy chain domain of the first arm is encoded by a first polypeptide chain; the first variable light chain domain of the first arm is encoded by a second polypeptide chain; the second variable heavy chain domain of the second arm is encoded by a third polypeptide chain; the second variable light chain domain of the second arm is encoded by a fourth polypeptide chain; and the first variable heavy chain domain and first variable light chain domain form a first PD-1 binding site and wherein the second variable heavy chain domain and second variable light chain domain form a second PD-1 binding site. In some embodiments, the first and second PD-1 binding sites are the same.

[0179] In some embodiments, the first and third polypeptide chain each further encode a hinge domain, a CH1 domain, and the Fc domain, and wherein the second and fourth polypeptide chain each further encode a CL domain. In some embodiments, the first and third polypeptide chains comprise the same sequence and the second and fourth polypeptide chains comprise the same sequence.

[0180] In some embodiments, the variable heavy and / or light chains comprise HCDR1-3 and / or LCDR1-3 as described herein for the anti-IL25 and anti-PD1 bispecific antibodies. In some embodiments, the variable heavy and / or light chains comprise VH and / or VL as described herein for the anti-IL25 and anti-PD1 bispecific antibodies.

[0181] In some embodiments, the first and second polypeptide chains are linked by one or more covalent disulfide bonds and the third and fourth polypeptide chains are linked by one or more covalent disulfide bonds. In some embodiments, the first and third polypeptide chains are linked by one or more covalent disulfide bonds.

[0182] In some embodiments, the subject has a solid tumor. In some embodiments, the cancer is hepatoma, Carcinoma of the prostate gland, renal cell carcinoma, hepatocellular carcinoma, colorectal carcinoma, prostate cancer, melanoma, colon, poeciliopsis lucida hepatocellular carcinoma, squamous cell carcinoma, bladder transitional cell carcinoma, colon adenocarcinoma, glioma, myeloid leukemia, neuroblastoma tumor, renal cortical adenocarcinoma, B cell lymphoma, lymphoma, plasmacytoma, pancreatic cancer, prostate cancer, or acute myeloid leukemia.

[0183] In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is ovarian cancer. In some embodiments, the subject has a hematopoietic malignancy. ActiveUS 209758784 79Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0184] In some embodiments, the tumor of the subject is reduced in volume. In some embodiments, growth of a tumor or cancer cells of the subject is inhibited. In some embodiments, immune-related adverse events are treated, reduced, or prevented in the subject.

[0185] In some embodiments, the immune-related adverse events comprise colitis, diarrhea, rash, pruritis, esophagitis, duodenitis, ileitis, neuritis, arthritis, vasculitis, nephritis, adrenal insufficiency, hepatitis, thrombocytopenia, anemia, pneumonitis, thyroiditis, hypophysitis, encephalitis, meningitis, uveitis, mucositis, rash, myocarditis, pericarditis, pancreatitis, colitis, enteritis, or any combination thereof.

[0186] In some embodiments, off-target immune infiltration of one or more untargeted organs in the subject is reduced or prevented. In some embodiments, CD3+T cells are not detected or are not present at elevated levels in one or more untargeted organs in the subject.

[0187] In some embodiments, the combination of the anti-IL-25 antibody or antigen binding fragment thereof and a PD-1 antibody, or antigen binding fragment thereof (e.g. antibodies / fragments thereof with variable regions or CDRs of SEQ ID NOs: 36-44 or SEQ ID NO: 76, 79) exhibits a synergistic effect on reducing a tumor volume, cancer treatment, or inhibiting tumor growth compared to the tumor volume reduction, cancer treatment effect, or tumor growth inhibition exhibited by administering a therapeutic dose of the anti-PD-1 antibody alone or a therapeutic dose of the anti-IL-25 antibody or antigen binding fragment thereof alone.

[0188] In some embodiments, the anti-IL-25 antibody or antigen binding fragment thereof and the PD-1 antibody, or antigen binding fragment thereof (e.g. antibodies / fragments thereof with variable regions or CDRs of SEQ ID NOs: 36-44 or SEQ ID NO: 76, 79) is administered concurrently as a single composition or as separate compositions. In some embodiments, the anti-IL-25 antibody or antigen binding fragment thereof and the PD-1 antibody, or antigen binding fragment thereof (e.g. antibodies / fragments thereof with variable regions or CDRs of SEQ ID NOs: 36-44 or SEQ ID NO: 76, 79) is administered sequentially.

[0189] In some embodiments, the subject is a human.

[0190] In certain aspects, the present disclosure provides a composition comprising therapeutically effect amounts of anti-IL-25 antibody or antigen binding fragment thereof and the PD-1 antibody, or antigen binding fragment thereof (e.g. antibodies / fragments thereof with variable regions or CDRs of SEQ ID NOs: 36-44 or SEQ ID NO: 76, 79). ActiveUS 209758784 80Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0191] In certain aspects, the present disclosure provides a combination in the form of a kit comprising two or more compositions, the first composition comprising a therapeutically effect amounts of anti-IL-25 antibody or antigen binding fragment thereof and the second composition comprising therapeutically effect amounts of an PD-1 antibody, or antigen binding fragment thereof (e.g. antibodies / fragments thereof with variable regions or CDRs of SEQ ID NOs: 36-44 or SEQ ID NO: 76, 79). In some embodiments, the compositions further comprise one or more pharmaceutically acceptable excipients. In some embodiments, the compositions further comprise a package insert or label providing directions for administering the compositions simultaneously, separately, or sequentially. Structure of Anti-PD-1 x Anti-IL-25 Antibodies

[0192] In some embodiments, the molecular three-dimensional structure of an anti-PD-1 x anti-IL-25 bispecific antibody can be predicted based on X-ray crystallography, and / or cryo-EM, and / or using structure prediction algorithms (e.g., machine learning algorithms) known in the art, such as AlphaFold or RaptorX. In some embodiments, the structure prediction algorithm is a computational method that is used to predict three-dimensional (3D) antibody structures based on a given nucleic acid or amino acid sequence. In some embodiments, the structure prediction algorithm predicts the 3D coordinates of all heavy atoms for a given antibody using a nucleic acid or amino acid sequence and / or aligned sequences of homologues as inputs. In some embodiments, the structure of an anti-PD-1 x anti-IL-25 bispecific antibody is predicted using a combination of methods, e.g., using a combination of AlphaFold (or any other structure prediction algorithm known in the art) and X-ray crystallography or cryo-EM. In some embodiments, the structure prediction is improved by combining the use of AlphaFold (or any other structure prediction algorithm known in the art) and X-ray crystallography or cryo-EM. In some embodiments, the structure of an anti-PD-1 x anti-IL-25 bispecific antibody is predicted by using a computational structure prediction algorithm (e.g., AlphaFold or RaptorX) and the structure prediction of the anti-PD-1 x anti-IL-25 bispecific antibody is then refined by using X-ray crystallography or cryo-EM. In some embodiments, the anti-PD-1 x anti-IL-25 bispecific antibody comprises a three-dimensional structure that is similar to the three-dimensional structure of an anti-PD-1 x anti-IL-25 bispecific antibody that comprises SEQ ID NO: 1 for the anti-IL-25 portion and SEQ ID NOs: 24 or 36-44 or SEQ ID NO: 76, 79 for the anti-PD-1 portion.

[0193] In some embodiments, the structure prediction algorithm can be used to model the structure of a first bispecific antibody (e.g., a reference anti-PD-1 x anti-IL-25 bispecific ActiveUS 209758784 81Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 antibody that comprises SEQ ID NO: 1 for the anti-IL-25 portion and SEQ ID NO: 24 or 36- 44 or SEQ ID NO: 76, 79 for the anti-PD-1 portion) and compare a predicted structure of second bispecific antibody (e.g., a second anti-PD-1 x anti-IL-25 bispecific antibody) against the predicted structure of the first antibody such that the second antibody can be categorized in the same class as the first bispecific antibody based on its structural similarity to the first bispecific antibody. In some embodiments, a metric of structural similarity between two antibodies can be obtained based on the output of a structure prediction algorithm known in the art. In some embodiments, the metric of structural similarity between two antibodies is based on a similarity distance.

[0194] In some embodiments, the structure of the anti-PD-1 x anti-IL-25 bispecific antibody allows the anti-PD-1 x anti-IL-25 bispecific antibody to bind to IL-25 and PD-1. In some embodiments, disclosed herein is a new class of anti-PD-1 x anti-IL-25 bispecific antibodies which comprise structural similarity to one another such that the new class of anti- PD-1 x anti-IL-25 bispecific antibodies are capable of binding to IL-25 and PD-1. In some embodiments, the anti-PD-1 x anti-IL-25 bispecific antibody comprises means for binding IL-25 and PD-1. In some embodiments, means for binding IL-25 and PD-1 comprises an anti-PD-1 x anti-IL-25. Compositions

[0195] In some embodiments, a prophylactic or therapeutic composition of this disclosure comprises one or more antibodies (or one or more polynucleotides encoding one or more antibodies) and is administered in a pharmaceutical composition that includes a pharmaceutically acceptable carrier. In some embodiments, the prophylactic or therapeutic composition is comprised of one or more antibodies (or one or more polynucleotides encoding one or more antibodies) comprising SEQ ID NOs: 1 and 24 (e.g., antibody “anti- SYB1.25” or “CEPO1.25”) or SEQ ID NOs: 1 and any of 36-44 or 76, 79. In some embodiments, the pharmaceutical composition is in the form of a spray, aerosol, gel, solution, emulsion, nanoparticle (e.g., lipid nanoparticle), or suspension.

[0196] The composition is preferably administered to a subject with a pharmaceutically acceptable carrier. Typically, in some embodiments, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation, which in some embodiments can render the formulation isotonic.

[0197] In certain embodiments, the one or more antibodies (or one or more polynucleotides encoding one or more antibodies) are provided as a composition comprising ActiveUS 209758784 82Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 any one of the antibodies described herein (e.g., anti-PD-1 x anti-IL-25 bispecific antibody) and a pharmaceutically acceptable carrier. In certain embodiments, the composition further comprises an adjuvant. In certain embodiments, the antibodies are conjugated with other molecules to increase their effectiveness as is known by those practiced in the art.

[0198] In some embodiments, the pharmaceutically acceptable carrier is selected from the group consisting of saline, Ringer's solution, dextrose solution, and a combination thereof. Other suitable pharmaceutically acceptable carriers known in the art are contemplated. Suitable carriers and their formulations are described in Remington's Pharmaceutical Sciences, 2005, Mack Publishing Co. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. The formulation may also comprise a lyophilized powder. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of antibodies being administered.

[0199] The phrase pharmaceutically acceptable carrier as used herein means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier is acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as butylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. The term carrier denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions ActiveUS 209758784 83Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 also are capable of being comingled with the compounds of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency. The composition may also include additional agents such as an isotonicity agent, a preservative, a surfactant, and, a divalent cation, preferably, zinc.

[0200] The composition can also include an excipient, or an agent for stabilization of an antibody composition, such as a buffer, a reducing agent, a bulk protein, amino acids (such as e.g., glycine or praline) or a carbohydrate. Typical carbohydrates useful in formulating compositions include but are not limited to sucrose, mannitol, lactose, trehalose, or glucose.

[0201] Surfactants may also be used to prevent soluble and insoluble aggregation and / or precipitation of antibodies included in the composition. Suitable surfactants include but are not limited to sorbitan trioleate, soya lecithin, and oleic acid. In certain cases, solution aerosols are preferred using solvents such as ethanol. Thus, formulations including antibodies can also include a surfactant that can reduce or prevent surface-induced aggregation of antibodies by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001% and 4% by weight of the formulation. In some embodiments, surfactants used with the present disclosure are polyoxyethylene sorbitan mono-oleate, polysorbate 80, polysorbate 20. Additional agents known in the art can also be included in the composition.

[0202] In some embodiments, the pharmaceutical compositions and dosage forms further comprise one or more compounds that reduce the rate by which an active ingredient will decay, or the composition will change in character. So called stabilizers or preservatives may include, but are not limited to, amino acids, antioxidants, pH buffers, or salt buffers. Nonlimiting examples of antioxidants include butylated hydroxy anisole (BHA), ascorbic acid and derivatives thereof, tocopherol and derivatives thereof, butylated hydroxy anisole and cysteine. Nonlimiting examples of preservatives include parabens, such as methyl or propyl p-hydroxybenzoate and benzalkonium chloride. Additional nonlimiting examples of amino acids include glycine or proline.

[0203] The present invention also teaches the stabilization (preventing or minimizing thermally or mechanically induced soluble or insoluble aggregation and / or precipitation of an inhibitor protein) of liquid solutions containing antibodies at neutral pH or less than neutral pH by the use of amino acids including proline or glycine, with or without divalent cations ActiveUS 209758784 84Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 resulting in clear or nearly clear solutions that are stable at room temperature or preferred for pharmaceutical administration.

[0204] In one embodiment, the composition is a pharmaceutical composition of single unit or multiple unit dosage forms. Pharmaceutical compositions of single unit or multiple unit dosage forms of the invention comprise a prophylactically or therapeutically effective amount of one or more compositions (e.g., a compound of the invention, or other prophylactic or therapeutic agent), typically, one or more vehicles, carriers, or excipients, stabilizing agents, and / or preservatives. Preferably, the vehicles, carriers, excipients, stabilizing agents and preservatives are pharmaceutically acceptable.

[0205] In some embodiments, the pharmaceutical compositions and dosage forms comprise anhydrous pharmaceutical compositions and dosage forms. Anhydrous pharmaceutical compositions and dosage forms of the invention can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions. Pharmaceutical compositions and dosage forms that comprise lactose and at least one active ingredient that comprise a primary or secondary amine are preferably anhydrous if substantial contact with moisture and / or humidity during manufacturing, packaging, and / or storage is expected. An anhydrous pharmaceutical composition should be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions are preferably packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastics, unit dose containers (e.g., vials), blister packs, and strip packs.

[0206] Suitable vehicles are well known to those skilled in the art of pharmacy, and non- limiting examples of suitable vehicles include glucose, sucrose, starch, lactose, gelatin, rice, silica gel, glycerol, talc, sodium chloride, dried skim milk, propylene glycol, water, sodium stearate, ethanol, and similar substances well known in the art. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid vehicles. Whether a particular vehicle is suitable for incorporation into a pharmaceutical composition or dosage form depends on a variety of factors well known in the art including, but not limited to, the way in which the dosage form will be administered to a patient and the specific active ingredients in the dosage form. Pharmaceutical vehicles can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. ActiveUS 209758784 85Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0207] The invention also provides that a pharmaceutical composition can be packaged in a hermetically sealed container such as an ampoule or sachette indicating the quantity. In one embodiment, the pharmaceutical composition can be supplied as a dry sterilized lyophilized powder in a delivery device suitable for administration to the lower airways of a patient. The pharmaceutical compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient. The pack can for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

[0208] Methods of preparing these formulations or compositions include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

[0209] Formulations of the invention suitable for administration may be in the form of powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as mouthwashes and the like, each containing a predetermined amount of a compound of the present invention (e.g., antibodies) as an active ingredient.

[0210] A liquid composition herein can be used as such with a delivery device, or they can be used for the preparation of pharmaceutically acceptable formulations comprising antibodies that are prepared for example by the method of spray drying. The methods of spray freeze-drying proteins for pharmaceutical administration disclosed in Maa et al., Curr. Pharm. Biotechnol., 2001, 1, 283-302, are incorporated herein. In another embodiment, the liquid solutions herein are freeze spray dried and the spray-dried product is collected as a dispersible peptide-containing powder that is therapeutically effective when administered to an individual.

[0211] The compounds and pharmaceutical compositions of the present invention can be employed in combination therapies, that is, the compounds and pharmaceutical compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures (e.g., antibodies can be used in combination treatment with another treatment such as antivirals or with a vaccine, and / or another treatment). The ActiveUS 209758784 86Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and / or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, the compound of the present invention may be administered concurrently with another therapeutic or prophylactic).

[0212] The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

[0213] The current invention provides for dosage forms comprising peptides suitable for treating cancer or other diseases. The dosage forms can be formulated, e.g., as sprays, aerosols, nanoparticles, liposomes, or other forms known to one of skill in the art. See, e.g., Remington's Pharmaceutical Sciences; Remington: The Science and Practice of Pharmacy supra; Pharmaceutical Dosage Forms and Drug Delivery Systems by Howard C., Ansel et al., Lippincott Williams & Wilkins; 7th edition (Oct.1, 1999).

[0214] Generally, a dosage form used in the acute treatment of a disease may contain larger amounts of one or more of the active ingredients it comprises than a dosage form used in the chronic treatment of the same disease. In addition, the prophylactically and therapeutically effective dosage form may vary among different conditions. For example, a therapeutically effective dosage form may contain one or more antibodies that have an appropriate therapeutic action when intending to treat cancer. On the other hand, a different effective dosage may contain one or more antibodies that have an appropriate prophylactic action when intending to prevent cancer. These and other ways in which specific dosage forms encompassed by this invention will vary from one another and will be readily apparent to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, 2005, Mack Publishing Co.; Remington: The Science and Practice of Pharmacy by Gennaro, Lippincott Williams & Wilkins; 20th edition (2003); Pharmaceutical Dosage Forms and Drug Delivery Systems by Howard C. Ansel et al., Lippincott Williams & Wilkins; 7th edition (Oct.1, 1999); and Encyclopedia of Pharmaceutical Technology, edited by Swarbrick, J. & J.C. ActiveUS 209758784 87Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 Boylan, Marcel Dekker, Inc., New York, 1988, which are incorporated herein by reference in their entirety.

[0215] The pH of a pharmaceutical composition or dosage form may also be adjusted to improve delivery and / or stability of one or more active ingredients. Similarly, the polarity of a solvent carrier, its ionic strength, or tonicity can be adjusted to improve delivery. Compounds such as stearates can also be added to pharmaceutical compositions or dosage forms to alter advantageously the hydrophilicity or lipophilicity of one or more active ingredients to improve delivery. In this regard, stearates can also serve as a lipid vehicle for the formulation, as an emulsifying agent or surfactant, and as a delivery enhancing or penetration-enhancing agent. Different salts, hydrates, or solvates of the active ingredients can be used to adjust further the properties of the resulting composition.

[0216] Compositions can be formulated with appropriate carriers and adjuvants using techniques to yield compositions suitable for prophylaxis or treatment. The compositions can include an adjuvant, such as, for example but not limited to, alum, poly IC, MF-59, squalene- based adjuvants, or liposomal based adjuvants suitable for prophylaxis or treatment.

[0217] In some embodiments, the antibodies described herein are encoded by nucleic acids which are prepared in a mRNA-LNP or a DNA-LNP formulation for administration to a subject. Antibody Production

[0218] The antibodies (e.g., bispecific antibodies) disclosed herein can be produced by any method known in the art. In some embodiments, the antibodies disclosed herein are produced by culturing a cell transfected or transformed with a vector comprising nucleic acid sequences encoding an antibody described herein and isolating the antibody. For example, the bispecific antibodies can be produced using chemical cross-linking of two IgG molecules, via fusion of two hybridomas, or via recombinant methods, e.g., via “knobs-into-holes” heterodimerization technology. See Marvin, Jonathan S., and Zhenping Zhu, Recombinant Approaches to IgG-like Bispecific Antibodies, Acta Pharmacologica Sinica 26.6 (2005): 649- 658, incorporated by reference in its entirety herein.

[0219] In some embodiments, antibodies are synthesized by the hybridoma culture method which results in antibodies that are not contaminated by other immunoglobulins. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal ActiveUS 209758784 88Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 antibodies to be used in accordance with the present invention may be made by a variety of techniques known in the art, including, for example, the hybridoma method (e.g., Kohler and Milstein., Nature, 256:495-97 (1975); Hongo et al, Hybridoma, 14 (3): 253-260 (1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling et al, in: Monoclonal Antibodies and T-CeIl Hybridomas 563-681 (Elsevier, N. Y., 1981)), recombinant DNA methods, phage-display technologies (see, e.g., Clackson et al, Nature, 352: 624-628 (1991); Marks et al, J. MoI Biol.222: 581-597 (1992); Sidhu et al, J. MoI Biol.338(2): 299-310 (2004); Lee et al, J. MoI Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. ScL USA 101(34): 12467-12472 (2004); and Lee et al, J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., Lonberg et al, Nature 368: 856- 859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al, Nature Biotechnol 14: 845-851 (1996); Neuberger, Nature Biotechnol.14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol.13: 65-93 (1995).

[0220] In some embodiments, expression of an antibody comprises expression vector(s) containing a polynucleotide that encodes an anti-PD-1 x anti-IL-25 bispecific antibody. Methods that are well known to those skilled in the art can be used to construct expression vectors comprising antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Particular embodiments provide replicable vectors comprising a nucleotide sequence encoding an anti-PD-1 x anti-IL- 25 bispecific antibody disclosed herein operably linked to a promoter. In preferred embodiments, such vectors may include a nucleotide sequence encoding the heavy chain of an antibody molecule (or fragment thereof), a nucleotide sequence encoding the light chain of an antibody (or fragment thereof), or both the heavy and light chain.

[0221] In some embodiments, a bispecific antibody described herein is made using the “knob-into-hole” or “KnH” technology. This method involves directing the pairing of two polypeptides together in vitro or in vivo by introducing a protuberance (knob) into one polypeptide and a cavity (hole) into the other polypeptide at an interface in which they interact. For example, KnHs have been introduced in the Fc:Fc binding interfaces, CL:CH1 interfaces or VH / VL interfaces of antibodies. See, e.g., US2007 / 0178552; WO 96 / 027011; WO 98 / 050431; Zhu et al. (1997) Protein Science 6:781-788, each of which are incorporated ActiveUS 209758784 89Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 in its entirety herein. For example, the production of bispecific antibodies with the KnHs strategy can be based on a single amino acid substitution in the opposite CH3 domains that promotes heavy chain heterodimerization. In some embodiments, in one of the heavy chains referred as a “knob” variant, a small amino acid has been replaced with a larger one in the CH3 domain. Subsequently, in the other heavy chain, a large amino acid has been replaced with a smaller one. A “hole” is formed, permitting the interaction with the “knobs.” See Ridgway JBB, Presta LG, Carter P., “Knobs-into-Holes” Engineering of Antibody CH3 Domains for Heavy Chain Heterodimerization. Protein Eng.1996, 9:617–621, incorporated in its entirety herein. This method can be used to pair two different heavy chains together during the manufacture of multispecific antibodies such as bispecific antibodies. For example, multispecific antibodies having KnH in their Fc regions can further comprise single variable domains linked to each Fc region, or further comprise different heavy chain variable domains that pair with similar or different light chain variable domains. KnH technology can also be used to pair two different receptor extracellular domains together or any other polypeptide sequences that comprises different target recognition sequences (e.g., including affibodies, peptibodies and other Fc fusions). In some embodiments, the bispecific antibodies described herein are scFv-Fc antibodies. In some embodiments, the scFv-Fc is a bispecific, bivalent molecule which is developed by fusing scFvs with different specificity to each Fc chain. In an scFv-Fc, the knobs-into-holes mutations in Fc force the formation of heterodimer.

[0222] The polynucleotide encoding the antibody may be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of the homologous murine sequences (U.S. Patent No.4,816,567; Morrison, et al, Proc. Natl Acad. ScL USA, 81:6851 (1984)), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art. Chimeric or hybrid antibodies also may be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. In some embodiments, an anti-IL- ActiveUS 209758784 90Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 25 and an anti-IL-PD1 are linkerd through a soluble cross-linker for example via their Fc domains.

[0223] Various expression systems for producing antibodies are known in the art, and include, prokaryotic (e.g., bacteria), plant, insect, yeast, and mammalian expression systems. Suitable cell lines, can be transformed, transduced, or transfected with nucleic acids containing coding sequences for antibodies or portions of antibodies disclosed herein in order to produce the antibody of interest. Expression vectors containing such nucleic acid sequences, which can be linked to at least one regulatory sequence in a manner that allows expression of the nucleotide sequence in a host cell, can be introduced via methods known in the art. Practitioners in the art understand that designing an expression vector can depend on factors, such as the choice of host cell to be transfected and / or the type and / or amount of desired protein to be expressed. Enhancer regions, which are those sequences found upstream or downstream of the promoter region in non-coding DNA regions, are also known in the art to be important in optimizing expression. If needed, origins of replication from viral sources can be employed, such as if a prokaryotic host is utilized for introduction of plasmid DNA. However, in eukaryotic organisms, chromosome integration is a common mechanism for DNA replication. For stable transfection of mammalian cells, a small fraction of cells can integrate introduced DNA into their genomes. The expression vector and transfection method utilized can be factors that contribute to a successful integration event. For stable amplification and expression of a desired protein, a vector containing DNA encoding a protein of interest (e.g., antibodies and fragments thereof) is stably integrated into the genome of eukaryotic cells (for example mammalian cells), resulting in the stable expression of transfected genes. A gene that encodes a selectable marker (for example, resistance to antibiotics or drugs) can be introduced into host cells along with the gene of interest in order to identify and select clones that stably express a gene encoding a protein of interest. Cells containing the gene of interest can be identified by drug selection wherein cells that have incorporated the selectable marker gene will survive in the presence of the drug. Cells that have not incorporated the gene for the selectable marker die. Surviving cells can then be screened for the production of the desired antibody molecule.

[0224] In some embodiments, the bispecific antibodies disclosed herein are encoded in a vector for expression in a cell line. In some embodiments, a first vector comprises a polynucleotide sequence that encodes an anti-IL-25 portion of a bispecific antibody, a second vector comprises a polynucleotide sequence that encodes an anti-PD1 portion of a bispecific ActiveUS 209758784 91Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 antibody, and each vector is transfected into one or more cell lines for expression. In some embodiments, a single vector comprises a polynucleotide sequence that encodes an anti-IL-25 portion of a bispecific antibody and an anti-PD1 portion of the bispecific antibody and the vector is transfected into one or more cell lines for expression. In some embodiments, one or more vectors comprise polynucleotide sequences encoding a light chain or a fragment thereof and a heavy chain or a fragment thereof the bispecific antibody. For example, in some embodiments, a first vector may comprise a polynucleotide sequence encoding a light chain, or fragment thereof, of an anti-IL-25 portion of the bispecific antibody, a second vector may comprise a polynucleotide sequence encoding a light chain, or fragment thereof, of an anti- PD1 portion of the bispecific antibody, a third vector may comprise a polynucleotide sequence encoding a heavy chain, or fragment thereof, of an anti-IL-25 portion of the bispecific antibody, and / or a fourth vector may comprise a polynucleotide sequence encoding a heavy chain, or fragment thereof, of an anti-PD1 portion of the bispecific antibody. In some embodiments, all four vectors are transfected into one or more cell lines for expression.

[0225] A host cell strain, which modulates the expression of the inserted sequences, or modifies and processes the nucleic acid in a specific fashion desired also may be chosen. Such modifications (for example, glycosylation and other post-translational modifications) and processing (for example, cleavage) of protein products may be important for the function of the antibody. Different host cell strains have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. As such, appropriate host systems or cell lines can be chosen to ensure the correct modification and processing of the foreign antibody expressed. Thus, eukaryotic host cells possessing the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.

[0226] Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art (Cleveland WL, et al., J Immunol Methods, 1983, 56(2): 221-234) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cell culturing conditions can vary according to the type of host cell selected. Commercially available media can be utilized.

[0227] Bispecific antibodies and antigen-binding fragments thereof disclosed herein can be purified from any human or non-human cell which expresses the antibody, including those ActiveUS 209758784 92Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 which have been transfected with expression constructs that express the antibody or fragments thereof. For antibody recovery, isolation and / or purification, the cell culture medium or cell lysate is centrifuged to remove particulate cells and cell debris. The desired antibody molecule is isolated or purified away from contaminating soluble proteins and polypeptides by suitable purification techniques. Non-limiting purification methods for proteins / antibodies include: size exclusion chromatography; affinity chromatography; ion exchange chromatography; ethanol precipitation; reverse phase HPLC; chromatography on a resin, such as silica, or cation exchange resin, e.g., DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, e.g., Sephadex G-75, Sepharose; protein A sepharose chromatography for removal of immunoglobulin contaminants; and the like. Other additives, such as protease inhibitors (e.g., PMSF or proteinase K) can be used to inhibit proteolytic degradation during purification. Purification procedures that can select for carbohydrates can also be used, e.g., ion-exchange soft gel chromatography, or HPLC using cation- or anion-exchange resins, in which the more acidic fraction(s) is / are collected. Dosage

[0228] A prophylactically effective or therapeutically effective amount is typically dependent on the weight of the subject being treated, the subject’s physical condition, the extensiveness of the condition to be treated, and the age of the subject being treated. In general, an anti-PD-1 x anti-IL-25 bispecific antibody, or polynucleotides encoding one or more antibodies, disclosed herein may be administered in an amount in the range of about 10 ng / kg body weight to about 100 mg / kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 50 μg / kg body weight to about 5 mg / kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 100 μg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 100 μg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.5 mg / kg body weight to about 20 mg / kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.5 mg / kg body weight to about 10 mg / kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 1 mg / kg body weight to about 5 mg / kg body weight per dose. In some embodiments, antibodies may be administered in an amount in the range of about 0.1 mg / kg body weight to about 0.5 mg / kg body weight per dose. In some embodiments, antibodies ActiveUS 209758784 93Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 may be administered in a dose of at least about 100 μg / kg body weight, at least about 250 μg / kg body weight, at least about 500 μg / kg body weight, at least about 750 μg / kg body weight, at least about 3 mg / kg body weight, at least about 5 mg / kg body weight, or at least about 10 mg / kg body weight.

[0229] In some methods, the dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 μg / mL or about 25-300 μg / mL. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of about 0.001 μg / mL to about 10 μg / mL. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of about 1 μg / mL to about 10 μg / mL. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of about 0.01 μg / mL to about 1 μg / mL. In some embodiments, the dosage is adjusted to achieve a plasma antibody concentration of about 0.01 μg / mL to about 0.1 μg / mL. Non-Limiting Embodiments

[0230] In certain aspects, the present disclosure provides a bispecific antibody or a fragment thereof, comprising: a first arm comprising a first variable heavy chain (VH) domain and a first variable light chain (VL) domain, wherein a portion of the first arm is capable of binding to a portion of an IL-25 protein; and a second arm comprising a second VH domain and a second VL domain, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

[0231] In some embodiments, the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together. In some embodiments, the first arm comprises a linker between the first VH domain and first VL domain. In some embodiments, the second arm comprises a linker between the first VH domain and first VL domain. In some embodiments, the linker is a glycine-serine linker.

[0232] In some embodiments, the first and second arms each further comprise a fragment, crystallizable (Fc) region. In some embodiments, the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa.

[0233] In some embodiments, the first VH domain comprises HCDR1 comprising SEQ ID NO: 5, HCDR2 comprising SEQ ID NO: 7, and HCDR3 comprising SEQ ID NO: 9 and wherein the first VL domain comprises LCDR1 comprising SEQ ID NO: 11, LCDR2 comprising SEQ ID NO: 13, and LCDR3 comprising SEQ ID NO: 15.

[0234] In some embodiments, the second VH domain comprises HCDR1 comprising SEQ ID NO: 28, HCDR2 comprising SEQ ID NO: 29, and HCDR3 comprising SEQ ID NO: ActiveUS 209758784 94Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 30 and wherein the first VL domain comprises LCDR1 comprising SEQ ID NO: 31, LCDR2 comprising SEQ ID NO: 32, and LCDR3 comprising SEQ ID NO: 33.

[0235] In some embodiments, the first VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 1 and wherein the first VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO: 1. In some embodiments, the second VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79 and wherein the second VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79.

[0236] In some embodiments, the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1 and wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1. In some embodiments, the second VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79 and wherein the second VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79. In some embodiments, the first arm comprises an amino acid sequence comprising SEQ ID NO: 1 and wherein the second arm comprises an amino acid sequence comprising SEQ ID NO: 24 or 36-44 or 76, 79.

[0237] In some embodiments, the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell. In some embodiments, the bispecific antibody is capable of enhancing T cell function.

[0238] In some embodiments, the PD-1 protein is located on a T cell, and wherein the bispecific antibody is capable of preventing the PD-1 protein from binding to a PDL-1 protein or a PDL-2 protein on a tumor cell.

[0239] In some embodiments, the bispecific antibody is capable of causing dephosphorylation of a tail of the PD-1 protein. In some embodiments, the bispecific antibody is capable of inducing a cytokine secretion in a T cell. In some embodiments, the cytokine secretion is a secretion of IL-2.

[0240] In some embodiments, the bispecific antibody does not induce immune-related adverse events. In some embodiments, the bispecific antibody does not induce off-target immune infiltration of one or more untargeted organs. ActiveUS 209758784 95Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0241] In certain aspects, the present disclosure provides a pharmaceutical composition comprising: any of the bispecific antibodies described above; and a pharmaceutically acceptable carrier.

[0242] In certain aspects, the present disclosure provides a method of preventing or treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition described above.

[0243] In some embodiments, the cancer is colorectal cancer, lung cancer, bladder cancer, breast cancer, cervical cancer, kidney cancer, leukemia, Hodgkin lymphoma, non- Hodgkin lymphoma, prostate cancer, skin cancer (e.g., melanoma), head and neck cancer, endometrial cancer, colon cancer, rectal cancer, liver cancer, thyroids cancer, esophageal cancer, renal cell cancer, or a combination thereof. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is lung cancer is small cell lung cancer. In some embodiments, the cancer is lung cancer is non-small cell lung cancer. In some embodiments, the cancer is bladder cancer. In some embodiments, the cancer is renal cell carcinoma.

[0244] In some embodiments, a tumor of the subject is reduced in volume. In some embodiments, growth of a tumor or cancer cells of the subject is inhibited.

[0245] In certain aspects, the present disclosure provides a method of preventing or treating autoimmunity in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition described above.

[0246] In certain aspects, the present disclosure provides a method of preventing or treating iRAEs in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition described above

[0247] In some embodiments, immune-related adverse events are treated, reduced, or prevented in the subject. In some embodiments, the immune-related adverse events comprise colitis, diarrhea, rash, pruritis, esophagitis, duodenitis, ileitis, neuritis, arthrhtis, vasculitis, nephritis, adrenal insufficiency, hepatitis, thrombocytopenia, anemia, pneumonitis, thyroiditis, hypophysitis, encephalitis, meningitis, uveitis, mucositis, rash, myocarditis, pericarditis, pancreatitis, colitis, enteritis, or any combination thereof. In some embodiments, off-target immune infiltration of one or more untargeted organs in the subject is reduced or prevented. In some embodiments, CD3+T cells are not detected or are not present at elevated levels in one or more untargeted organs in the subject.

[0248] In some embodiments, treatment with the composition described above has a synergistic effect on reducing a tumor volume, cancer treatment, or inhibiting tumor growth ActiveUS 209758784 96Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 compared to the tumor volume reduction, cancer treatment effect, or tumor growth inhibition exhibited by administering a therapeutic dose of the one or more anti-PD1 antibodies or antigen binding fragment thereof alone or a therapeutic dose of the anti-IL-25 antibody or antigen binding fragment thereof alone.

[0249] In some embodiments, the subject is a human.

[0250] In certain aspects, the present disclosure provides a kit for generating a bispecific antibody or fragment thereof, the kit comprising one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described above.

[0251] In certain aspects, the present disclosure provides a kit for generating a bispecific antibody or fragment thereof, the kit comprising: a first vector comprising a polynucleotide sequence encoding a first arm of the bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a IL-25 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

[0252] In some embodiments, the first vector and the second vector are the same vector. In some embodiments, the first vector and the second vector are two different vectors.

[0253] In some embodiments, the first arm comprises a first VH domain and a first VL domain and the second arm comprises a second VH domain and a second VL domain, wherein a VH domain of the first arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 12, wherein a first VL domain of the first arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, wherein a VH domain of the second arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79, and wherein a VL domain of the second arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79.

[0254] In certain aspects, the present disclosure provides one or more host cells comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described above.

[0255] In certain aspects, the present disclosure provides a ne or more host cells comprising: a first vector comprising a polynucleotide sequence encoding a first arm of a bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of an IL-25 protein; and a second vector comprising a polynucleotide ActiveUS 209758784 97Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

[0256] In some embodiments, the first vector and the second vector are the same vector. In some embodiments, the first vector and the second vector are two different vectors.

[0257] In some embodiments, the first arm comprises a first VH domain and a first VL domain and the second arm comprises a second VH domain and a second VL domain, wherein the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, wherein a VH domain of the second arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79, and wherein a VL domain of the second arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79.

[0258] In certain aspects, the present disclosure provides a method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells described above under conditions suitable for an expression of the one or more vectors; and recovering the bispecific antibody or fragment thereof.

[0259] In certain aspects, the present disclosure provides a method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells described above under conditions suitable for an expression of the first vector and the second vector; and recovering the bispecific antibody or fragment thereof.

[0260] In certain aspects, the present disclosure provides a composition comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies described above.

[0261] In certain aspects, the present disclosure provides a composition comprising: a first vector comprising a polynucleotide sequence encoding a first arm of the bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a IL-25 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

[0262] In some embodiments, the first vector and the second vector are the same vector. In some embodiments, the first vector and the second vector are two different vectors.

[0263] In some embodiments, the first arm comprises a first VH domain and a first VL domain, wherein the second arm comprises a second VH domain and a second VL domain, ActiveUS 209758784 98Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 wherein the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, wherein a VH domain of the second arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79, and wherein a VL domain of the second arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79.

[0264] In certain aspects, the present disclosure provides a means for binding: a portion of a IL-25 protein; and a portion of a PD-1 protein. In some embodiments, the means comprises a bispecific antibody or fragment thereof.

[0265] In some embodiments, the bispecific antibody or a fragment thereof comprises: a first arm comprising a first VH domain and a first VL domain, wherein a portion of the first arm is capable of binding to the portion of the IL-25 protein; and a second arm comprising a second VH domain and a second VL domain, wherein a portion of the second arm is capable of binding to the portion of the PD-1 protein.

[0266] In some embodiments, the first VH domain comprises HCDR1 comprising SEQ ID NO: 5, HCDR2 comprising SEQ ID NO: 7, and HCDR3 comprising SEQ ID NO: 9 and wherein the first VL domain comprises LCDR1 comprising SEQ ID NO: 11, LCDR2 comprising SEQ ID NO: 13, and LCDR3 comprising SEQ ID NO: 15. In some embodiments, the second VH domain comprises HCDR1 comprising SEQ ID NO: 28, HCDR2 comprising SEQ ID NO: 29, and HCDR3 comprising SEQ ID NO: 30 and wherein the first VL domain comprises LCDR1 comprising SEQ ID NO: 31, LCDR2 comprising SEQ ID NO: 32, and LCDR3 comprising SEQ ID NO: 33.

[0267] In some embodiments, the first VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 1 and wherein the first VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO: 1. In some embodiments, the second VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79 and wherein the second VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79. ActiveUS 209758784 99Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0268] In some embodiments, the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, and wherein the portion of the first arm is capable of binding to the portion of the IL-25 protein. In some embodiments, the second VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79, wherein the second VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79, and wherein the portion of second arm is capable of binding to the portion of the PD-1 protein. In some embodiments, the first arm comprises an amino acid sequence comprising SEQ ID NO: 1, wherein the second arm comprises an amino acid sequence comprising SEQ ID NO: 24 or 36-44 or 76, 79.

[0269] In some embodiments, the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell. In some embodiments, the bispecific antibody is capable of enhancing T cell function.

[0270] In some embodiments, the bispecific antibody is capable of binding to the IL-25 portion of the IL-25 protein and to the PD-1 portion on the PD-1 protein, wherein the IL-25 protein and PD-1 protein are located on a same T cell.

[0271] In some embodiments, the bispecific antibody is capable of disrupting a downstream signaling of a PD-1 mediated response in a T cell. In some embodiments, the bispecific antibody is capable of enhancing T cell function.

[0272] In some embodiments, PD-1 protein is located on a T cell, and wherein the bispecific antibody is capable of preventing the PD-1 protein from binding to a PDL-1 protein or a PDL-2 protein on a tumor cell.

[0273] In some embodiments, the bispecific antibody is capable of causing dephosphorylation of a tail of the PD-1 protein. In some embodiments, the bispecific antibody is capable of inducing a cytokine secretion in a T cell. In some embodiments, the cytokine secretion is a secretion of IL-2.

[0274] In some embodiments, the bispecific antibody does not induce immune-related adverse events. In some embodiments, the bispecific antibody does not induce off-target immune infiltration of one or more untargeted organs.

[0275] In some embodiments, the bispecific antibody reduces off-target cytokine distribution in one or more untargeted organs. In some embodiments, the bispecific antibody reduces off-target cytokine distribution by neutralizing the one or more cytokines. In some embodiments, the cytokine with reduced distribution is IL-25. In some embodiments, the ActiveUS 209758784 100Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 untargeted organ with reduced cytokine distribution is the lung. In some embodiments, the untargeted organ with reduced cytokine distribution is the liver. In some embodiments, the untargeted organ with reduced cytokine distribution is the brain. EXAMPLES

[0276] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results. EXAMPLE 1 - Anti-PD-1 x IL-25 bispecific antibody as novel cancer immunotherapy

[0277] CEPO1.25, a novel anti-PD-1 x anti-IL-25 bispecific antibody was generated. In vivo experiments validating the tumor-suppressing efficacy of CEPO1.25 was performed. B6 mice were inoculated with MC38 mouse colon adenocarcinoma cells. Once tumor size reached ~50mm^3, mice were treated with CEPO1.25 through intraperitoneal injections twice a week for two weeks at 50μg, 100μg or 200μg per dose. Recordings of tumor measurements were graphed, and the anti-tumor efficacy of CEPO1.25 was validated. Mice responded to CEPO1.25 in a dose-responsive manner, and CEPO1.25 enhanced the survival of the mice.

[0278] Patients will benefit from the novel anti-PD-1 x anti-IL-25 bispecific antibody by its increased antitumor efficacy, survival, and reduced risk of irAEs. There are 1.9 million new cancer cases annually in the U.S., and about 43.6% of the patients are eligible for ICI treatment. In some embodiments, the cancer to be treated is lung cancer, including small cell carcinoma (SCLAC) and non-small cell lung cancer (NSCLC). In some embodiments, the cancer to be treated is renal cell carcinoma (RCC), or bladder cancer. In 2023, about 238,340 patients were diagnosed with lung cancer. Current FDA-approved ICIs in the U.S. market are Opdivo®, Keytruda®, Yervoy®, Tecentriq®, Bavencio®, and Imfinzi®. There are currently no approved next-generation bispecific ICIs in the U.S. Multiple bispecific antibodies against PD-1 / PD-L1 and other targets are currently in development, ranging from preclinical to phase II clinical trials, however, none of them targets IL-25 or aims to resolve immune toxicity as described herein.

[0279] Described herein are experiments designed to test these bispecific antibodies in vivo using syngeneic tumor model, in comparison to no treatment. ActiveUS 209758784 101Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0280] CEPO1.25 demonstrates very promising results and uses anti-mouse PD-1 to enable testing in B6 mice. To optimize and humanize the antibody, the anti-PD-1 arm of the antibody is replaced with anti-human PD-1 sequences. Furthermore, new anti-human IL-25 sequences are used. To test the modified antibody's anti-tumor efficacy and immune toxicity in vivo, transgenic B6 mice with human PD-1 receptors (B6 / hPD1) are used. Anti-human IL- 25 cross-reacts with mouse IL-25 due to high sequence homology. A lead clone of anti- human PD-1 x anti-human IL-25 bispecific antibody will be selected and tested for its dose- response and superiority over the standard anti-PD-1 monoclonal antibody in terms of anti- tumor efficacy and immune safety. Detailed plan is as follows:

[0281] Discover new anti-IL-25 clones: Five clones of monospecific anti-IL-25 antibodies will be generated using the single-B antibody discovery. Surface Plasmon Resonance (SPR) will be performed to measure the affinity of the anti-IL-25 clones.

[0282] Design and produce new clones of CEPO1.25: Three of five clones will be selected based on their affinities for bispecific antibody design and production.

[0283] Select lead clone in-vivo: The three newly generated clones of anti-PD-1 x anti- IL-25 antibodies will be compared in vivo. To do that, B6 / hPD1 mice will be inoculated with cells of MC38 (mouse colon adenocarcinoma). Once tumor size reaches 50mm^3, the mice will be treated with bispecific aPD1 x aIL25 antibodies twice a week for three weeks at 200μg per dose. The experiment will include six mice for each bispecific antibody clones and six untreated mice (24 mice total). Tumors will be measured, and tumor growth will be assessed. Organs (liver, lung, heart, pancreas, tumors) will be collected at the endpoint, and histology will be performed to assess immune toxicity across organs. Based on the results, a clone of the aPD1 x aIL-25 antibody will be selected as the lead candidate.

[0284] Upscale lead production: The production of the selected clone of aPD1 x aIL-25 antibody will be upscaled and an anti-PD1 control antibody will be produced.

[0285] Efficacy experiment: The dose-response and the superiority over the PD-1 monoclonal antibody of the optimized CEPO1.25 in B6 / hPD1 will be validated, assessing antitumor efficacy and immune toxicity. Like the previous in vivo experiment, B6 / hPD1 will be inoculated with MC38 cells. Once tumor size reaches 50mm^3, the mice will be treated twice a week with bispecific aPD1 x aIL25 antibodies at 100μg, 200μg or 400μg per dose or with aPD1 monoclonal antibody at 200μg per dose. The experiment will include six mice for each experimental group and six untreated mice (30 mice total). Tumors will be measured, ActiveUS 209758784 102Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 and tumor growth will be assessed. Organs will be collected at the endpoint, and histology will be performed to assess immune toxicity across organs. EXAMPLE 2 - In vitro binding of anti-PD-1 Abs

[0286] As shown in FIG.4A-B, binding curves to human-PD-1-His-coated plates was quantified by ELISA. FIG.4A shows binding curves for anti-PD1 antibody clones 01, 02, 03, and 07, anti-human PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgG1 isotype control, and blank. FIG.4B shows binding curves for anti-PD1 antibody clones 09, 51, 55, 79, and 80, anti-human PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgG1 isotype control, and blank.

[0287] Table shows EC50 values for anti-PD1 antibody clones 01, 02, 03, and 07, anti- human PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgG1 isotype control, and blank. Table 5 shows EC50 values for anti-PD1 antibody clones 09, 51, 55, 79, and 80, anti- human PD-1 antibody Penbio (pembrolizumab), anti-HEL-human IgG1 isotype control, and blank. EC50 values were calculated with GraphPad Prism (v10.2.1). These results show that the anti-PD1 antibody clones are capable of binding to PD-1, in some cases with lower or similar EC50 values when compared to the commercially available pembrolizumab.

[0288] FIG.5 shows binding of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, and 80 to cell-expressed PD-1 by flow cytometry. Jurkat T cells stably overexpressing human-PD-1 ActiveUS 209758784 103Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 were incubated with 10 µg / ml (FIG.5 left) or 1 µg / ml (FIG.5 right) of each anti-PD-1 clone on ice for 30 minutes, washed twice, and then incubated for 30 minutes on ice with a fluorescent anti-human-Fc secondary antibody. Following two additional washes, cells were analyzed on a BD LSRFortessaTMflow cytometer. These results show that the anti-PD-1 clones bind to cell-expressed PD-1.

[0289] FIG.6 shows anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, and 80 blocking the binding of rhPD-L2 to cell-expressed PD-1. Jurkat T cells stably overexpressing human-PD- 1 were incubated with 10 µg / ml of each anti-PD-1 clone on ice for 30 minutes, washed twice, and then incubated for 1 hour on ice with 0.5 µg / ml (FIG.14 left) or 5 µg / ml (FIG.14 right) of recombinant human PD-L2 (with mouse-Fc). Following two additional washes, the cells were incubated for 30 minutes on ice with a fluorescent anti-mouse-Fc secondary antibody, washed twice, and analyzed on a BD LSRFortessaTMflow cytometer. These results show that the anti-PD-1 clones block the binding of rhPD-L2 to cell-expressed PD-1.

[0290] FIG.7 shows IL-2 concentrations following addition of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 and SEE (Staphylococcal Enterotoxin E) in Jurkat-Raji co- culture compared to no SEE and no antibody control and SEE and no antibody control. Jurkat T cells stably overexpressing human-PD-1 and Raji B cells stably overexpressing human-PD-L1 were co-cultured for 24 hours in the presence of 10 µg / ml of each anti-PD-1 clone and 100 pg / ml SEE (Staphylococcal Enterotoxin E). IL-2 concentrations in the supernatants were determined by ELISA. These results show that anti-PD-1 clones can increase IL-2 concentrations in the supernatants compared to no antibody controls.

[0291] FIG.8 shows IL-2 concentrations determined by ELISA following addition of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 at different concentrations (10, 2, 0.5 or 0.1 µg / ml) and SEE (Staphylococcal Enterotoxin E) in Jurkat-Raji co-culture compared to no SEE and no antibody control and SEE and no antibody control. Jurkat T cells stably overexpressing human-PD-1 and Raji B cells stably overexpressing human-PD-L1 were co- cultured for 24 hours in the presence of different concentrations (10, 2, 0.5 or 0.1 µg / ml) of each anti-PD-1 clone and 100 pg / ml SEE (Staphylococcal Enterotoxin E). These results show that anti-PD-1 clones can increase IL-2 concentrations in a dose dependent manner in the supernatants compared to no antibody controls.

[0292] FIG.9 shows concentrations of IL-2, IFNγ, IL-6, IL-4 and IL-1β determined by ELISA in PBMCs in the presence of anti-PD-1 clones 01, 02, 03, 07, 09, 51, 55, 79, or 80 and SEE (Staphylococcal Enterotoxin E). PBMCs from 3 healthy donors were incubated for ActiveUS 209758784 104Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 24 hours in the presence of 10 µg / ml of each anti-PD-1 clone and 100 pg / ml SEE (Staphylococcal Enterotoxin E). The concentrations of IL-2, IFNγ, IL-6, IL-4 and IL-1β in the supernatants were determined by ELISA. These results show that anti-PD-1 clones can increase IL-2, IFNγ, IL-6, IL-4 and IL-1β concentrations in the supernatants compared to no antibody controls. EXAMPLE 3- Anti-PD-1 x IL-25 bispecific antibody as novel cancer immunotherapy

[0293] In 2023, the U.S. reported 1.9 million new cancer cases, resulting in over 600,000 fatalities. Cancer immunotherapy, which utilizes the immune system to combat cancer, represents the cutting edge of cancer treatment. This includes various forms such as immune checkpoint inhibitors (ICIs), CAR-T cells, engineered TCRs, tumor-infiltrating lymphocytes, and therapeutic cancer vaccines. Among these, ICIs are the least invasive, most broadly applicable, and most cost-efficient option. Of the 1.9 million newly diagnosed cancer patients in 2023, 810,000 are candidates for ICI treatments.

[0294] ICIs are recognized as the new standard of care for numerous cancer types, enhancing patient survival and outcomes. Nevertheless, the current response rate to ICIs is only 12.5%, highlighting a critical need for more effective ICIs to treat tumors. Additionally, ICIs can cause immune-related adverse events (irAEs), some of which—like myocarditis— may pose serious risks to life. Furthermore, severe irAEs require the discontinuation of ICI therapy, which diminishes the overall effectiveness and applicability of these potentially life- saving treatments. Therefore, a significant demand is for a next-generation ICI that offers superior tumor suppression while minimizing immune-related toxicities.

[0295] We developed SYB1.25, a new bispecific antibody targeting anti-PD-1 and anti- IL-25. We validated its tumor-suppressing effects in three preclinical mouse models: MC38 colon cancer, B16F10 melanoma, and EO771 breast cancer. In the colon cancer study, B6 mice received MC38 mouse colon adenocarcinoma cells. When the tumors reached approximately 50mm^3, the mice were administered SYB1.25 via intraperitoneal injections twice weekly over two weeks, at 50µl, 100µg, or 200µg. Tumor size measurements were recorded and graphed, confirming the anti-tumor effectiveness of SYB1.25. Mice exhibited a dose-dependent response to SYB1.25, leading to improved survival rates. For the melanoma model, B6 mice were injected with B16F10 cells. Upon tumors growing to about 30mm^3, treatment options included aPD1, aPD1+aIL25 combination, SYB1.25, aPDL1xIL25, or no treatment at all. Results indicated that SYB1.25 exhibited superior anti-tumor activity among the group. In the third model, we utilized EO771 breast tumor cells and swapped B6 WT for ActiveUS 209758784 105Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 aged B6 Lpr mice susceptible to irAEs. In this experiment, the treatments administered were aPD1, aPD1+aIL25, SYB1.25, or none. This trial confirmed that SYB1.25 had the highest anti-tumor efficacy and demonstrated that mice receiving SYB1.25 did not experience irAEs, while those treated with aPD1 suffered from severe irAEs.

[0296] In the U.S., the FDA has approved several immune checkpoint inhibitors (ICIs), including Opdivo®, Keytruda®, Yervoy®, Tecentriq®, Bavencio®, and Imfinzi®. However, there are currently no approved next-generation bispecific ICIs available in the U.S. While bispecific antibodies that target PD-1 / PD-L1 and other pathways are under development, these bispecifics target immune checkpoints and cytokines simultaneously. Nonetheless, none specifically target IL-25 or tackle immune toxicity like SYB1.25 does.

[0297] FIG.11 shows that Nivolumab / Pembrolizumab bound CD4+ T-cells in cancer patients with irAE-arthritis express high IL-17RA, which is the receptor for IL25 cytokine.

[0298] CEPO1.25, a novel anti-PD-1 x anti-IL-25 bispecific antibody was generated (FIG 12).

[0299] ELISA assays in FIG.13 show that CEPO1.25 shows higher affinity to both PD-1 and IL-25 compared to an anti-PD1 antibody. Furthermore, flow cytometry in FIG.14 shows better binding of CEPO1.25 to the surface of T-Cells relative to pembrolizumab.

[0300] FIG.14 shows differential regulation of cytokines produced by PBMCs when treated with CEPO1.25. Human PBMCs are pre-activated with 1000pg / ml SEE. Treatment with CEPO1.25 shows higher levels of IFN^ and IL-2 when compared to control, anti-IL-25 treatment, or co-treatment with anti-PD-1 and anti-IL-25. In addition, treatment with CEPO1.25 shows significantly less IL-6 levels when compared to anti-IL-25 treatment or co- treatment with anti-PD-1 and anti-IL-25.

[0301] FIG.15 shows preliminary testing of the CEPO1.25 antibody. B6 mice were inoculated with MC38 mouse colon adenocarcinoma cells. Four days post inoculation, mice were treated with CEPO1.25 through intraperitoneal injections on Day 7 and Day 11 at 0μg or 200μg per dose. Recordings of tumor measurements were graphed over 22 days, and the anti-tumor efficacy of CEPO1.25 was validated. CEPO1.25 inhibited tumor growth in mice.

[0302] FIG.16 shows in vivo experiments validating the tumor-suppressing efficacy of CEPO1.25. B6 mice were inoculated with MC38 mouse colon adenocarcinoma cells. Seven days post inoculation, mice were treated with CEPO1.25 through intraperitoneal injections twice a week for two weeks at 0μg, 50μg, 100μg or 200μg per dose. Recordings of tumor measurements were graphed over 25 days, and the anti-tumor efficacy of CEPO1.25 was ActiveUS 209758784 106Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 validated. Mice responded to CEPO1.25 in a dose-responsive manner, and CEPO1.25 enhanced the survival of the mice.

[0303] FIG.17 shows that CEPO1.25 did not show the same tumor suppressing effect in a similar mouse model. B6 LpR mice were inoculated with MC38 mouse colon adenocarcinoma cells. On Days 0, 4, 8, and 12, mice were treated with either an anti-PD1 antibody or CEPO1.25 through intraperitoneal injections at 200μg per dose. Recordings of tumor measurements were graphed over 15 days. Interestingly, CEPO 1.25 had no significant effect on tumor growth.

[0304] FIG.18 shows that CEPO1.25 is capable of suppressing breast tumor progression in an irAE mouse model. Aged B6 LpR mice were inoculated with EO771 mouse breast cancer cells. On Days 0, 3, 10, 13, 17, and 20, mice were treated with no antibody, 100μg of an anti-PD1 antibody, or 150μg of CEPO1.25 through intraperitoneal injections. Recordings of tumor measurements were graphed over 22 days, and the anti-tumor efficacy of CEPO1.25 was validated. Mice treated with CEPO1.25 had lower primary tumor volume when compared to control.

[0305] FIG.19 shows that CEPO1.25 is capable of suppressing breast tumor progression more effectively than a combination of anti-PD-1 and anti-IL-25 treatment. Aged B6 LpR mice were inoculated with EO771 mouse breast cancer cells. On Days 0, 3, 10, 13, 17, and 20, mice were treated with no antibody, 100μg of an anti-PD1 antibody and 100μg of an anti- IL25 antibody, or 150μg of CEPO1.25 through intraperitoneal injections. Recordings of tumor measurements were graphed, and the anti-tumor efficacy of CEPO1.25 was validated. Mice treated with CEPO1.25 had significantly lower primary tumor volume when compared to the combination treatment.

[0306] After 22 days of treatment, the livers and lungs of the mice from FIGS.9-10 were H&E stained and visualized for immune infiltration (FIG.20). H&E reveals lower immune infiltration severity of both the liver and lung of mice treated with CEPO1.25 and the IL- 25 / anti-PD1 combination therapy when compared to either the untreated or anti-PD1 treated samples.

[0307] Sections from the tumor, liver, and lungs of the mice from FIGS.9-10 were also immunofluorescent stained for IgG (FIG.21). The IL-25 / anti-PD1 combination therapy shows stronger IgG signal in the tumor compared to the untreated, anti-PD-1, or CEPO1.25 treatment. ActiveUS 209758784 107Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025

[0308] B6 wildtype females were inoculated with 500,000 B16F10 tumor cells (FIG.22). At day 6 post inoculation, tumor volumes were approximately 25mm3and treatments were initiated. Per treatment, mice were dosed with nothing, 100µg aPD1+100µg aIL25 (200µg total), or 150µg of aPD1xIL25 bispecific antibody through IP injection. Treatments were given every 3 days for four times total. At 21 days post tumor inoculation, the mice were euthanized. The tumor growth curve shows the average tumor volume of each experiment group over the course of day 6 to day 21 post tumor cell inoculation.

[0309] B6 wildtype females were inoculated with 500,000 B16F10 tumor cells (FIG.23). At day 6 post inoculation, tumor volumes were approximately 25mm3and treatments were initiated. Per treatment, mice were dosed with nothing or 150µg of aPDL1xIL25 bispecific antibody through IP injection. Treatments were given every 3 days for four times total. At 21 days post tumor inoculation, the mice were euthanized. The tumor growth curve shows the average tumor volume of each experiment group over the course of day 6 to day 21 post tumor cell inoculation. The bispecific antibody CEPO1.25 can be humanized by substituting the anti-PD-1 arm with anti-human PD-1 sequences described herein and / or substituting the anti-IL-25 arm with anti-human IL-25 sequences. Bispecific antibodies are evaluated for anti-tumor effectiveness and immune toxicity in vivo, for example, using transgenic B6 mice that express human PD-1 receptors (BD / hPD1). Anti-human IL-25 arms will cross-react with mouse IL-25 due to sequence similarity. In some embodiments, the bispecific antibody comprises the Fc region of IgG1. In some embodiments, the bispecific antibody comprises the Fc region of IgG4.

[0310] Antibody clones are tested in vitro using the PBMC and Raji-Jurkat assays. Cytokine secretion levels (including IL2, IL6, IFNg, IL1b) is measured by ELISA.

[0311] Anti-PD-1 x anti-IL-25 are tested in vivo using the MC38 model. B6 / hPD1 mice are inoculated with MC38 cells (mouse colon adenocarcinoma). Once the tumor size reaches 50 mm³, the mice are treated with bispecific aPD1xaIL25 antibodies twice a week for two weeks at 200 µg per dose. The experiment includes ten mice treated with the bispecific antibody and ten untreated mice. Tumors are measured, and tumor growth is assessed.

[0312] Anti-PD-1 x anti-IL-25 antibodies are compared in vivo using the B16F10 model. To do this, B6 / hPD1 mice are inoculated with cells from B16F10 (mouse melanoma). Once the tumor size reaches 50 mm3, the mice are treated with bispecific aPD1xaIL25 antibodies twice a week for two weeks at 200 µg per dose. The experiment includes ten mice treated ActiveUS 209758784 108Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 with the bispecific antibody and ten untreated mice. Tumors are measured, and tumor growth is assessed. Example 4- In vivo treatment with aPD1xaIL25 (SYB1.25) antibody

[0313] Treatment with SYB1.25 increase IL-2 secretion and effector CD8+ CD69+ T cells and decreases IL-6 secretion (FIGS.24-25).

[0314] SYB 1.25 decreases MC38 colon tumor volume in B6 mice (FIG.26).

[0315] SYB 1.25 decreases EO771 breast tumor volume in B6 mice (FIGS.27A-B).

[0316] SYB 1.25 decreases B16F10 melanoma tumor volume in B6 mice (FIG.28).

[0317] SYB 1.25 decreases CT26 colon tumor volume in B6 mice and keeps tumor volume down after rechallenge (FIG.29-30).

[0318] SYB 1.25 decreases off-target immune severity in liver and lung compared to Pembrolizumab or anti-PD-1 and anti-IL-25 cotreatment (FIGS.31-32) ActiveUS 209758784 109

Claims

Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 CLAIMS What is claimed is:

1. A bispecific antibody or a fragment thereof, comprising: a first arm comprising a first variable heavy chain (VH) domain and a first variable light chain (VL) domain, wherein a portion of the first arm is capable of binding to a portion of an IL-25 protein; and a second arm comprising a second VH domain and a second VL domain, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

2. The bispecific antibody of claim 1, wherein the first arm is encoded by a first polypeptide chain and the second arm is encoded by a second polypeptide chain that associate together.

3. The bispecific antibody of claims 1-2, wherein the first arm comprises a linker between the first VH domain and first VL domain.

4. The bispecific antibody of claims 1-3, wherein the second arm comprises a linker between the first VH domain and first VL domain.

5. The bispecific antibody of claims 3-4, wherein the linker is a glycine-serine linker.

6. The bispecific antibody of claim 2, wherein the first and second arms each further comprise a fragment, crystallizable (Fc) region.

7. The bispecific antibody of claim 6, wherein the Fc region of the first arm comprises knob mutations and the Fc region of the second arm comprise hole mutations, or vice versa.

8. The bispecific antibody of claims 1-7, wherein the first VH domain comprises HCDR1 comprising SEQ ID NO: 5, HCDR2 comprising SEQ ID NO: 7, and HCDR3 comprising SEQ ID NO: 9 and wherein the first VL domain comprises LCDR1 comprising SEQ ID NO: 11, LCDR2 comprising SEQ ID NO: 13, and LCDR3 comprising SEQ ID NO:

15.

9. The bispecific antibody of claims 1-8, wherein the second VH domain comprises HCDR1 comprising SEQ ID NO: 28, HCDR2 comprising SEQ ID NO: 29, and HCDR3 ActiveUS 209758784 110Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 comprising SEQ ID NO: 30 and wherein the second VL domain comprises LCDR1 comprising SEQ ID NO: 31, LCDR2 comprising SEQ ID NO: 32, and LCDR3 comprising SEQ ID NO: 33 or wherein the second VH domain comprises HCDR1, HCDR2, and HCDR3 of Figure 10 or Table 1 and wherein the second VL domain comprises LCDR1, LCDR2, and LCDR3 of Figure 10 or Table 3.

10. The bispecific antibody of claims 1-9, wherein the first VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 1 and wherein the first VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO:

1.

11. The bispecific antibody of claims 1-10, wherein the second VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79 and wherein the second VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79.

12. The bispecific antibody of claims 1-11, wherein the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1 and wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO:

1.

13. The bispecific antibody of claims 1-12, wherein the second VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79 and wherein the second VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79.

14. The bispecific antibody of claims 1-13, wherein the first arm comprises an amino acid sequence comprising SEQ ID NO: 1 and wherein the second arm comprises an amino acid sequence comprising SEQ ID NO: 24 or 36-44 or 76, 79.

15. The bispecific antibody of claims 1-14, wherein the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell. ActiveUS 209758784 111Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 16. The bispecific antibody of claims 1-15, wherein the bispecific antibody is capable of enhancing T cell function.

17. The bispecific antibody of claims 1-15, wherein the PD-1 protein is located on a T cell, and wherein the bispecific antibody is capable of preventing the PD-1 protein from binding to a PDL-1 protein or a PDL-2 protein on a tumor cell.

18. The bispecific antibody of claim 17, wherein the bispecific antibody is capable of causing dephosphorylation of a tail of the PD-1 protein.

19. The bispecific antibody of claim 17, wherein the bispecific antibody is capable of inducing a cytokine secretion in a T cell.

20. The bispecific antibody of claim 19, wherein the cytokine secretion is a secretion of IL-2.

21. The bispecific antibody of claims 1-15, wherein the bispecific antibody does not induce immune-related adverse events.

22. The bispecific antibody of claims 1-15, wherein the bispecific antibody does not induce off-target immune infiltration of one or more untargeted organs.

23. A pharmaceutical composition comprising: the bispecific antibody of any of claims 1- 22; and a pharmaceutically acceptable carrier.

24. A method of preventing or treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition of claim 23.

25. The method of claim 24, wherein the cancer is colorectal cancer, lung cancer, bladder cancer, breast cancer, cervical cancer, kidney cancer, leukemia, Hodgkin lymphoma, non- Hodgkin lymphoma, prostate cancer, skin cancer (e.g., melanoma), head and neck cancer, endometrial cancer, colon cancer, rectal cancer, liver cancer, thyroids cancer, esophageal cancer, renal cell cancer, or a combination thereof.

26. The method of claim 24, wherein the cancer is lung cancer.

27. The method of claim 26, wherein the cancer is lung cancer is small cell lung cancer. ActiveUS 209758784 112Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 28. The method of claim 26, wherein the cancer is lung cancer is non-small cell lung cancer.

29. The method of claim 24, wherein the cancer is bladder cancer.

30. The method of claim 24, wherein the cancer is renal cell carcinoma.

31. The method of claims 24-30, wherein a tumor of the subject is reduced in volume.

32. The method of claims 24-30, wherein growth of a tumor or cancer cells of the subject is inhibited.

33. The method of claims 24-30, wherein immune-related adverse events are treated, reduced, or prevented in the subject.

34. The method of claim 33, wherein the immune-related adverse events comprise colitis, diarrhea, rash, pruritis, esophagitis, duodenitis, ileitis, neuritis, arthritis, vasculitis, nephritis, adrenal insufficiency, hepatitis, thrombocytopenia, anemia, pneumonitis, thyroiditis, hypophysitis, encephalitis, meningitis, uveitis, mucositis, rash, myocarditis, pericarditis, pancreatitis, colitis, enteritis, or any combination thereof.

35. The method of claims 24-34, wherein off-target immune infiltration of one or more untargeted organs in the subject is reduced or prevented.

36. The method of claims 24-34, wherein CD3+T cells are not detected or are not present at elevated levels in one or more untargeted organs in the subject.

37. The method of claims 24-36, wherein treatment with the composition of claim 23 has a synergistic effect on reducing a tumor volume, cancer treatment, or inhibiting tumor growth compared to the tumor volume reduction, cancer treatment effect, or tumor growth inhibition exhibited by administering a therapeutic dose of the one or more anti-PD1 antibodies or antigen binding fragment thereof alone or a therapeutic dose of the anti-IL-25 antibody or antigen binding fragment thereof alone.

38. The method of claims 34-37, wherein the subject is a human. ActiveUS 209758784 113Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 39. A kit for generating a bispecific antibody or fragment thereof, the kit comprising one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies of any of claims 1-22.

40. A kit for generating a bispecific antibody or fragment thereof, the kit comprising: a first vector comprising a polynucleotide sequence encoding a first arm of the bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a IL-25 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

41. The kit of claim 40, wherein the first vector and the second vector are the same vector.

42. The kit of claim 40, wherein the first vector and the second vector are two different vectors.

43. The kit of claims 40-42, wherein the first arm comprises a first VH domain and a first VL domain and the second arm comprises a second VH domain and a second VL domain, wherein a VH domain of the first arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein a first VL domain of the first arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, wherein a VH domain of the second arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79, and wherein a VL domain of the second arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79.

44. One or more host cells comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies of any of claims 1-22.

45. One or more host cells comprising: a first vector comprising a polynucleotide sequence encoding a first arm of a bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of an IL-25 protein; and ActiveUS 209758784 114Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

46. The one or more host cells of claim 45, wherein the first vector and the second vector are the same vector.

47. The one or more host cells of claim 45, wherein the first vector and the second vector are two different vectors.

48. The one or more host cells of claim 45-47, wherein the first arm comprises a first VH domain and a first VL domain and the second arm comprises a second VH domain and a second VL domain, wherein the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, wherein a VH domain of the second arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79, and wherein a VL domain of the second arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79.

49. A method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells of claim 44 under conditions suitable for an expression of the one or more vectors; and recovering the bispecific antibody or fragment thereof.

50. A method of making a bispecific antibody or fragment thereof comprising: culturing the one or more host cells of any of claims 45-48 under conditions suitable for an expression of the first vector and the second vector; and recovering the bispecific antibody or fragment thereof.

51. A composition comprising: one or more vectors comprising a polynucleotide sequence encoding any of the bispecific antibodies of any of claims 1-22.

52. A composition comprising: ActiveUS 209758784 115Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 a first vector comprising a polynucleotide sequence encoding a first arm of the bispecific antibody or fragment thereof, wherein a portion of the first arm is capable of binding to a portion of a IL-25 protein; and a second vector comprising a polynucleotide sequence encoding a second arm of the bispecific antibody or fragment thereof, wherein a portion of the second arm is capable of binding to a portion of a PD-1 protein.

53. The composition of claim 52, wherein the first vector and the second vector are the same vector.

54. The composition of claim 52, wherein the first vector and the second vector are two different vectors.

55. The composition of claim 52-54, wherein the first arm comprises a first VH domain and a first VL domain, wherein the second arm comprises a second VH domain and a second VL domain, wherein the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, wherein a VH domain of the second arm comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79, and wherein a VL domain of the second arm comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79.

56. A means for binding: a portion of a IL-25 protein; and a portion of a PD-1 protein.

57. The means of claim 56, wherein the means comprises a bispecific antibody or fragment thereof.

58. The means of claim 57, wherein the bispecific antibody or a fragment thereof comprises: a first arm comprising a first VH domain and a first VL domain, wherein a portion of the first arm is capable of binding to the portion of the IL-25 protein; and ActiveUS 209758784 116Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 a second arm comprising a second VH domain and a second VL domain, wherein a portion of the second arm is capable of binding to the portion of the PD-1 protein.

59. The means of claim 58, wherein the first VH domain comprises HCDR1 comprising SEQ ID NO: 5, HCDR2 comprising SEQ ID NO: 7, and HCDR3 comprising SEQ ID NO: 9 and wherein the first VL domain comprises LCDR1 comprising SEQ ID NO: 11, LCDR2 comprising SEQ ID NO: 13, and LCDR3 comprising SEQ ID NO:

15.

60. The bispecific antibody of claims 58-59, wherein the second VH domain comprises HCDR1 comprising SEQ ID NO: 28, HCDR2 comprising SEQ ID NO: 29, and HCDR3 comprising SEQ ID NO: 30 and wherein the second VL domain comprises LCDR1 comprising SEQ ID NO: 31, LCDR2 comprising SEQ ID NO: 32, and LCDR3 comprising SEQ ID NO: 33 or wherein the second VH domain comprises HCDR1, HCDR2, and HCDR3 of Figure 10 or Table 1 and wherein the second VL domain comprises LCDR1, LCDR2, and LCDR3 of Figure 10 or Table 3.

61. The bispecific antibody of claims 58-60, wherein the first VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 1 and wherein the first VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO:

1.

62. The bispecific antibody of claims 58-61, wherein the second VH domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79 and wherein the second VL domain comprises an amino acid sequence 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 ,95, 96, 97, 98, or 99% identical to the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79.

63. The means of claim 58, wherein the first VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 1, wherein the first VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 1, and wherein the portion of the first arm is capable of binding to the portion of the IL-25 protein.

64. The means of claims 58 or 63, wherein the second VH domain comprises an amino acid sequence of the VH portion of SEQ ID NO: 24 or 36-44 or 76, 79, wherein the second ActiveUS 209758784 117Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 VL domain comprises an amino acid sequence of the VL portion of SEQ ID NO: 24 or 36-44 or 76, 79, and wherein the portion of second arm is capable of binding to the portion of the PD-1 protein.

65. The means of claim 58, wherein the first arm comprises an amino acid sequence comprising SEQ ID NO: 1, wherein the second arm comprises an amino acid sequence comprising SEQ ID NO: 24 or 36-44 or 76, 79.

66. The means of claims 56-65, wherein the bispecific antibody is capable of disrupting downstream signaling of a PD-1 mediated response in a T cell.

67. The means of claims 56-65, wherein the bispecific antibody is capable of enhancing T cell function.

68. The means of claims 56-65, wherein the bispecific antibody is capable of binding to the IL-25 portion of the IL-25 protein and to the PD-1 portion on the PD-1 protein, wherein the IL-25 protein and PD-1 protein are located on a same T cell.

69. The means of claims 56-65, wherein the bispecific antibody is capable of disrupting a downstream signaling of a PD-1 mediated response in a T cell.

70. The means of claims 56-65, wherein the bispecific antibody is capable of enhancing T cell function.

71. The means of claims 56-65, wherein the PD-1 protein is located on a T cell, and wherein the bispecific antibody is capable of preventing the PD-1 protein from binding to a PDL-1 protein or a PDL-2 protein on a tumor cell.

72. The means of claims 56-65, wherein the bispecific antibody is capable of causing dephosphorylation of a tail of the PD-1 protein.

73. The means of claims 56-65, wherein the bispecific antibody is capable of inducing a cytokine secretion in a T cell.

74. The means of claim 73, wherein the cytokine secretion is a secretion of IL-2.

75. The means of claims 56-65, wherein the bispecific antibody does not induce immune- related adverse events. ActiveUS 209758784 118Attorney Docket No.: 0019240.01340WO1 Date of Electronic Filing: May 20, 2025 76. The means of claims 56-65, wherein the bispecific antibody does not induce off-target immune infiltration of one or more untargeted organs.

77. A method of preventing or treating cancer in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the composition of claim 23.

78. The method of claim 77, wherein the cancer is breast cancer.

79. The method of claim 77, wherein the composition does not induce immune-related adverse events. ActiveUS 209758784 119

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